CA2672205A1

CA2672205A1 - Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides

Info

Publication number: CA2672205A1
Application number: CA002672205A
Authority: CA
Inventors: Zhenwei Miao; Junjie Liu
Original assignee: Individual
Current assignee: Ambrx Inc
Priority date: 2006-12-18
Filing date: 2007-12-18
Publication date: 2008-06-26
Also published as: JP2010512800A; MX2009006617A; WO2011028195A3; CN101578264A; AU2007333737A1; EP2079687A1; WO2008077079A1; WO2011028195A2; KR20090100407A

Abstract

Disclosed herein are non-natural amino acids and polypeptides that include at least one non-natural amino acid, and methods for making such non-natural amino acids and polypeptides. The non-natural amino acids, by themselves or as a part of a polypeptide, can include a wide range of functionalities, but typically have at least one indole, carbonyl, and/or hydrazine group. Also disclosed herein are non-natural amino acid polypeptides that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such polypeptides. Typically, the modified non-natural amino acid polypeptides include at least one indole, carbonyl, and/or hydrazine group. Further disclosed are methods for using such non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, including therapeutic, diagnostic, and other biotechnology uses.

Description

DEMANDE OU BREVET VOLUMINEUX

LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

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VOLUME

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COMPOSITIONS CONTAINING, METHODS INVOLVING, AND USES OF NON-NATURAL
AMINO ACIDS AND POLYPEPTIDES

RELATED APPLICATIONS

[00011 This application claims the benefit of U.S. Non-Provisional Patent Application No. 60/870,594 filed December 18, 2006.

FIELD OF THE INVENTION

100021 Non-natural amino acids, polypeptides containing at least one non-natural amino acid, methods for producing such. non-natural amino,acids and polypeptides, and uses of such non-natural amino acids and polypeptides for diagnostic, environmental, industrial, and therapeutic uses.

BACKGROUND OF THE INVENTION

(00031 The ability to incorporate non-genetically,encoded amino acids (i.e., "non-natural amino acids") into proteins permits the introduction of chemical functional groups that could provide valuable alternatives to the naturally-occurring functional groups, such as the epsilon -NH2 of lysine, the sulthydryl -SH of cysteine, the imino group of histidine, etc. Certain cheniical functional groups are documented as inert to the functional groups found in the 20 common, genetically-encoded amino acids but react cleanly and efficiently to form stable linkages with fttnctional groups that can be incorporated onto non-natural amino acids.
100041 Methods are now available to selectively introduce chemical functional groups that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 comnion, genetically-encoded amino acids and that may be used to react efficiently and selectively with reagents comprising certain functional groups to form stable covalent linkages.

SUMMARY OF THE INVENTION

100051 Described herein are methods, conipositions, techniques and strategies for making, purifying, characterizing, and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides. In one aspect are methods, compositions, techniques and strategies for derivatizing.a non-natural amino acid and/or a non-natural amino acid polypeptide. In one, embodiment, such methods, compositions, techniques and strategies involve chemical derivatization, in other embodiments, biological derivatization, in other embodiments, physical derivatizationõin other embodiments a cornbination of derivatizations. In further or additional embodiments, such derivatizations are regioselective. In further or additional embodiments, such derivatizations are regiospecific. In further or additional embodiments, such dcrivations are stoichiometric or near stoichiometric in both the non-natural amino acid containing reagent and the derivitizing reagent. In further or additional embodiments, such derivatizations are rapid at ambient temperature. In further or additional embodiments, such derivatizations occur in aqueous solutions. In further or additional embodiments, such derivatizations occur at a pH between about 4 and about 10. In further or additional embodiments, such derivatizations are stoichiometric, near stoichiometric or stoichiometric -like in both the non-natural amino acid containing reagent and the derivatizing reagent. In further or additional embodiments are provided methods which allow the stoichiometric, near stoichiometric or stoichiometric-like incorporation of a desired group onto a non-natural amino acid polypeptide. In further or additional I

embodinients are provided strategies, "reaction mixtures, synthetic conditions-which allow the stoichiometric, near stoichiometric or stoichiometric -like incorporation of a desired group onto a non-natural 'amino acid polypeptide.
[00061 In one aspect are non-natural aniino acids for the chemical derivatization of peptides and proteins based'upon the reactivity of a carbonyl group or masked carbonyl group, including a group containing.at least one.ketone;group, and/or at least one aldehyde groups. In further or additional aspects are non-natural amino acids for the chemical derivatization of peptides and proteins based upon the reactivity ofa hydrazine group or masked hydrazine group. In further or additional embodiments, at least one of the aforementioned non-natural amino acids is incorporated into a polypeptide, that is, such embodiments are non-natural amino acid polypeptides. In further or additional embodiments, the non-natural amino acids are functionalized on their sidechains such that their reaction with a derivatizing molecule generates an indole containing linkage. In further or additional embodiments are non-natural amino acid polypeptides that can react with a derivatizing niolecule to generate a non-natural amino acid polypeptide containing an indole linkage. In further or additional embodinients, the non-natural amino acids are selected from amino acids having carbonyl and/or hydrazine sidechains. In further or additional embodimeints, the non-nathiral amino acids comprise a masked sidechain, including a masked hydrazine group and/or a masked carbonyl group.
100071 In further or additional embodiments, the non-natural aniino. acids comprise carbonyl sidechains where the carbonyl is selected from a ketone or an aldehyde. In another embodiment:are non-natural amino acids containing a functional group that is capable of forming an indole upon treatment with an appropriately functionalized reagent. In some embodiments are non-natural amino acids containing an indole moiety. In a further embodiment are-non=natural amino acids containing an indole-moiety for the treatment of disorders, conditions or diseases. In a further or additional embodinient, the non-natural amino acids resemble a natural amino aci`d in structure but contains one. of the aforementioned functional groups. In another or further embodinient the non-natural amino acids resemble phenylalanine or. tyrosine (aromatic amino acids); while in a separate embodiment, the non-natural amino acid's resemble alanine and leucine (hydrophobic amino acids). In onc embodiment, the non-natural amino acids have properties that are distinct from those of the natural amino acids. In one embodiment, such distinct properties are. the chemical reactivity of the side chain, in a further embodiment this distinct chemical reactivity permits the side chain of the non-natural amino acid to undergo a reaction while being a unit of a polypeptide even though the side chains of the naturally-occurring amino acid units in the same polypeptide do not undergo the aforementioned reaction. In a further embodiment, the side chain of the non-natural amino acid has chemistry orthogonal to those of the naturally-occurring amino acids. In a further embodiment, the side chain-of the non-natural anuno acid,comprises an electrophile-containing inoiety;
in a further embodiment, the electrophile-containing moiety on.the sidechain of the non-natural amino acid can undergo, nucleophilic attack to generate, a heterocycle-derivatized protein, including a nitrogen-containing heterocycle-derivatized protein (e.g., containing an indole moiety). In any ofthe aforementioned ernbodiments in this paragraph, the.non-natural amino=acid are either separate molecules or incorporated into a polypeptide of any length; if thelatter, then the polypeptide, in.some:embodiments, further incorporates.additional naturally-occurring or non-natural amino acids.

100081 In another aspect are hydrazine-substituted molecules for the production of derivatized non-natural anlino acid polypeptides based upon an indole-containing heterocycle linkage.
In a further embodiment are hydrazine-substituted molecules used to derivatize carbonyl-containing non-natural amino acid polypeptides via the formation ofan indole-containing:heterocycle linkage. In furtherembodiments the aforementioned carbonyl-S containing non-natural amino acid polypeptides are ketone-containing non=natural.amino acid polypeptides. In further or additional embodiments, the carbonyl-containing non-natural aniino acids, comprise sidechains where the carbonyl.is selected from a ketone, or an aldehyde. In further or additional embodiments, the hydrazine-substituted molecules comprise a. desired functionality: In further or additional embodiments,,the hydrazine-substitutedmolecules are hydrazine-substituted polyethylene glycol (PEG).molecules. In a further embodiment, the sidechain of the non-natural anuno acid has:a chetrustry orthogonal to those of the naturally-occurring amino acids that allows the non-natural anuno acid to. reacC selective,ly with the hydrazine-substituted molecules. In a further embodiment, the sidechain of the non-natural amino acid corriprises an electrophile-containing moiety that reacts selectively with the hydrazine-containing. molecule; in a further embodiment, the electrophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack to generate a heterocycle-derivatized protein, including a nitrogen-containing heterocycle-derivatized protein. In a further aspect related to the embodiments described in this paragraph are the modified non-natural amino acid polypeptides that result from the reaction of the derivatizing molecule with the non-natural amino acid polypeptides. Further embodiments include any further modifications of the already modified inon-natural arnino acid polypeptides.
100091 In another aspect are carbonyl-substituted molecules for the production of derivatized non-natural amino. acid polypepti,des based upon a heterocycle, including. a nitrogen-containing heterocycle (e.g., an indole or a multi-cyclic structure containing an indole portion), linkage. In a further embodiment are carbonyl-substituted molecules used to derivatize hydrazine-containingnon-natural amino acid polypeptides via. the formation of an indole-containfng heterocycle linkage. In a-further embodiment are carbonyl-substituted molecules that can form such heterocycle tivith a hydrazine-containing non-natural amino acid potypeptide in a pH range between about 1 and about 6. In a further embodiment are carbonyl-substituted. molecules used to derivatize hydrazine-containing non-natural amino acid polypeptid.es via the formation of an indole-containing heterocycle linkage between the" derivatizing molecule and the hydrazine-containing non-natural amino acid polypeptides. In a ftirther embodiment the carbonyl-substituted molecules are ketone-substitued molecules, in otlter aspects aldehyde-substituted molecules. In further embodiments, the carbonyl-substituted molecules comprise a desired functionality. ln further or additional embodiments, the aldehyde-substituted molecules are aldeltyde-substituted polyethylene glycol (PEG) molecules. In a furtlier enibodiment, the sidechain of the non-natural amino acid has a chetnistry orthogonal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react selectively with the carbonyl-substituted molccules. In a further embodiment, the sidechain of tlie non-natural amino acid comprises a moiety (e.g., hydrazine group) that reacts selectively witli the carbonyl-containing molecule; in a further cmbodiment, the nucleophilic moiety on the sidechain of the non-natural amino acid can undergo electrophilic attack to generate a heterocyclic-derivatized proteui, including a nitrogen-containing heterocycle-derivatized protein. In a further aspect related to the embodiments described in this paragraph are the modified non-natural amino acid polypeptides that result from the reaction of the derivatizing molecule with the non-natural aniino acid.polypeptides. Further embodiments include any further modificatioris of the already modified non-natural amino acid polyp,eptides.

100101 In another aspect are inono-, bi- and multi-funct9onal linkers for the generatioit of derivatized non-natural anvno acid "polypeptides based upon an indole-containing heterocycle linkage. In one embodiment are molecular linkers (bi- and niulti-funetional) that can be used to. connect carbonyl-containing non-natural amino acid polypeptides to other molecules. In another embodiment are molecular linkers (bi- and niulti-functional) that can be used to connect hydrazine-containing non-natural anrino acid polypeptides to other molecules. In another embodiment.. the carbonyl-containing non-natural amino acid polypeptides comprise a ketone, or an aldehyde. In an embodinient utilizing a hydrazine-containing non-natural amino acid polypeptide, the molecular linker contains a carbonyl group at one of its termini; in further embodiments, the carbonyl group is selected from an aldehyde group, or a ketone group. In further or additional embodiments, the hydrazine-substituted linker molecules are hydrazine-substituted polyethylene glycol (PEG) linker molecules. In fiutlter or additional embodiments; the: carbonyl-substituted linker molecules are carbonyl=substituted polyethylene glycol (PEG) linker molecules. In further cmbodiments, the phrase "other molecules"
includes, by way of example only;
proteins, other polymers and small molecules. In further or. additional embodiments, the hydrazine=containing.
inolecular linkers comprise the same orequivalent groups on all termini'so that. upon reaction with a carbonyl-containing non-natural amino acid polypep6de, the xesulting product is the homo-niultimerization of the carbonyl-containing non-natural amino acidpolypeptide. In further embodiments, the homo-multimerization is a homo-dimerization. In further or additional embodiments, the carbonyl-containing molecular linkers comprise the same or equivalent groups on all termini so that upon reaction with a hydrazine-containing non-natural amino acid polypeptide, the resulting product is the homo-multimerization of the hydrazine-containing non-natural amino acid polypeptide. In further embodiments, the homo-multimerization is a homo-dimerization. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occutring amino acids that allows the non-natural amino acid to react selectively with the hydrazine-substituted linker molecules. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occuning amino acids that allows the non-natural amino acid to react selectively with the carbonyl-substituted linker molecules. In a further embodiment, the sidechain of the non-natural amino acid comprises an electrophile-containing moiety that reacts selectively with the hydrazine-containing linker molecule; -in a further embodiment; the electrophile-containing moiety on the sidechain of the non-iiatural aniino acid can undergo,:nucleophilic attack by the hydrazine-containing linker molecule to generate a heterocycle-derivatized protein, including a nitrogen-containing heterocycle-derivatized protein. In a further aspect related to, the embodiments described im this paragraph are the linked (modified) non-natural amino acid polypeptides that resul.t from the reaction of the, linker-.molecule with the;
non-natural. amino acid polypeptides.
Further embodiments include=any-further modifications of the alreadylinked (iiiodified) non-natural amino acid polypeptides.
100111 In one aspect~are methods to derivatize proteins via the reaction of carbonyl and hydrazine reactants to generate a heterocycle-derivatized protein, including a nitrogen-containing.
heterocycle-derivatized protein.
included within this aspect are methods for the derivatization of proteins based upon the condensation of carbonyl- and hydrazine-containing reactants to generate a heterocycle-derivatized protein adduct, including a nitrogen-containing heterocycle-derivatized protein adduct. In additional or further embodiments are methods to derivatize ketone-containing proteins or aldehyde-containing proteins with hydrazine-functionalized polyethylene glycol (PEG) molecules. In yet additional or further aspects, the hydrazine-substituted molecule can include proteins, other polymers, and small molecules.

100121 In another aspect are methods for the chemical synthesis of hydrazine-substituted molecules for the derivatization of carbonyl-substituted proteins. In, one embodiment, the hydrazine-substituted molecule can comprise peptides, other polymers (non-branched and branched) and small niolecules. In one embodiment are methods for the preparation of hydrazine-substituted molecules suitable for the derivatization of carbonyl-containing non-natural amino acid polypeptides, including by way of example only, ketone-, or aldehyde-containing non-natural amino acid polypeptides. In a further or additional embodiment, the non-natural amino acids arc incorporated site-specifically during the in vivo translation of proteins. In a further or additional embodiment, the hydrazine-substituted molecules allow for the site-specific derivatization of carbonyl-containing non-natural amino acids via nucleophilic attack of each carbonyl group to form a heterocycle-derivatized polypeptide, including a nitrogen-containing heterocycle-derivatized polypeptide in a site-specific fashion. In a further or additional entbodiment, the method for the preparation of hydrazine-substituted molecules provides access to a wide variety of site-specifically derivatized polypeptides. In a further or additional embodiment are methods for synthesizing hydrazine-functionalized polyethyleneglycol (PEG) molecules.
100131 In another aspect are methods for the chemical synthesis of carbonyl-substituted molecules for the derivatization.of hydrazine-substituted non-natural amino acid polypeptides.
In one embodiment, the carbonvl-substituted niolecule is a ketone-, and/or an aldehyde-substituted molecule.
In another embodiment, the carbonyl-substituted molecules include proteins, polymers (non-branched and branched) and small molecules.
In a further or additional embodiment, such methods complement technology that enables the site-specific incorporation of non-iiatural amino acids during the in vivo translation of proteins. In a further or additional embodiment are general methods for the preparation of carbonyl-substituted molecules suitable for reaction with hydrazine-containing non-natural amino acid polypeptides to provide site-specifically derivatized non-natural amino acid polypeptides. In a further or additional embodiment are methods for synthesizing carbonyl-substituted polyethylene glycol (PEG) molecules.
100141 In another aspect are methods for the chemical derivatization of carbonyl-substituted non-natural amino acid polypeptides using a liydrazine-containing bi-functional linker. In one embodiment are methods for attaching a hydrazine-substituted linker to a carbonyl-substituted protein via a condensation reaction to generate a heterocycle, including a nitrogen-containing heterocycle, linkage. In further or additional embodiments, the carbonyl-substituted non-natural amino acid is a ketone-, and/or an aldehyde-substituted non-natural amino acid. In further or additional embodiments, the non-natural amino acid polypeptides are derivatized site-specifically and/or with precise control of three-dimensional structure, using a hydrazine-containing bi-functional linker. In one embodiment, such methods are used to attach motecular linkers (mono- bi- and multi-functional) to carbonyl-containing (including by way of example ketone-, and/or an aldehyde-containing) non-natural amino acid polypeptides, wherein at least one of the linker termini contains a hydrazine group which can link to the carbonyl-containing rion-natural amino acid polypeptides via a heterocycle, including a nitrogen-containing heterocycle, linkage. In a further or additional embodiment, these linkers are used to connect the carbonyl-containing non-natural amino acid polypeptides- to other molecules,, including by way of example, proteins, other polymers (branched and non-branched) and small molecules.

[00151 In some embodiments, the non-natural amino acid polypeptide is linked to a water soluble polymer. In some embodiments, the water soluble polymer comprises a polyethylene glycol moiety. In some embodiments, the polyethylene glycol molecule is a bifunctional polymer. In some embodiments, the bifunctional polymer is linked to a second polypeptide. In some embodiments, the second polypeptide is identical to the first polypeptide, in other embodiments, the second polypeptide is a different polypeptide. In some embodiments, the non-natural amino acid polypeptide comprises at least two amino acids. linked to a water soluble polymer comprising a poly(ethylene glycol) moiety.
100161 In some embodiments, the non-natural anuno acid polypeptide comprises a substitution, addition or deletion that increases af6nity of the non-natural amino acid polypeptide for a receptor. In some embodiments, the non-natural amino acid polypeptide comprises a substitution, addition, or deletion that increases the stability of the non-natural amino acid polypeptide. In some embodiments, the non-natural amino acid polypeptide comprises a substitution, addition, or deletion that increases the aqueous solubility of the non-natural amino acid polypeptide. In some embodiments, the non-natural amino acid polypeptide comprises a substitution, addition, or deletion that increases the solubility of the non-natural amino acid polypeptide produced in a host cell. In some embodiments, the non-natura] amino acid polypeptide comprises a substitution, addition, or deletion that modulates protease resistance, serum half-life, immunogenicity, and/or expression relative to the amino-acid polypeptide without the substitution, addition or deletion.
100I7J In some embodiments, the non-natural amino acid polypeptide is an agonist, partial agonist, antagonist, partial antagonist, or inverse agonist. In some embodiments, the agonist, partial agonist, antagonist, partial antagonist, or inverse agonist comprises a non-natural amino acid linked to a water soluble polymer. In some enibodinients, the water polymer comprises a polyethylene glycol moiery. In some embodiments, the polypeptide comprising a non-natural amino acid linked to a water soluble polymer prevents dimerization of the corresponding receptor. In some embodiments, the polypeptide contprising a non-natural amino acid linked to a water soluble polymer modulates binding of the polypeptide to a binding partner, ligand or receptor. In some embodiments, the polypeptide comprising a non-natural amino acid linked to a water soluble polymer modulates one or more properties or activities of the polypeptide.
100181 ln some embodiments, the selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon.
100191 Also described herein are methods of making a non-natural amino acid polypeptide litilced to a water solublc polymer. In some embodiments, the niethod comprises contacting an isolated polypeptide comprising a non-natural amino acid with a water soluble polymer comprising a moiety that reacts with the non-natural amino acid. In some embodiments, the non-natural amino acid incorporated into is reactive toward a water soluble polymer that is otherwise unreactive toward any of the 20 conunon amino acids. In some embodiments, the water polymer comprises a polyethylene glycol moiety. The molecular weight of the polymer optionally is within a desired polymer molecular weight range.
100201 Also described herein are compositions comprising a polypeptide comprising at least one of the non-natural amino acids described herein and a pharmaceutically acceptable canrier. In some embodiments, the non-natural amino acid is linked to a water soluble polymer. Also described herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a polypeptide, wherein at least one amino acid is substituted by a non-natural aniino acid. In some embodiments, the non-natural amino acid comprises a saccharide moiety. In sonie embodiments, the water soluble polymer is linked to the polypeptide via a saccharide moiety. Also described herein are prodrugs of the non-naturalamino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides; further described herein are compositions coniprising such prodrugs and a pharmaceutically acceptable carrier. Also described herein are metabolites of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides; in some embodiments, such metabolites have a desired activity that complements or synergizes with the activity of the non-natural amino acids, non-natural aniino acid polypeptides, and modified non-natural amino acid polypeptides. Also described herein are the use of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides described herein to provide a desired metabolite to an organism, including a patient in need of sucli metabolite.
100211 Also described herein are cells comprising a polynucleotide encod'uig the polypeptide comprising a selector codon. In some embodinients, the cells comprise_an orthogonal RNA
synthetase, and/or an orthogonal tRNA for substitutinga,non-natural amino acid into the polypeptide. In some,embodiments the cells are in a cell culture, whereas in other embodiments the cells of part of a multicellular organism, including amphibians, reptiles, birds, and mamnials. In any of the cell embodiments, further enibodiments include expression of the polynucleotide to produce the.non-natural amino acid polypeptide. In other embodiments are organisms that can utilize the non-natural amino acids described herein to produce a non-natural amino acid polypeptide, including a modified non-natural amino acid polypeptide. In other embodiments are organisms containing the non-natural anuno acids, the non-natural amino acid polypeptides, and/or the modified non-natural amino acid polypeptides described herein. Such organisms include unicellular and multicellular organisms, including amphibians, reptiles, birds, and niammals. In some embodiments, the non-natural amino acid polypeptide is produced in vitro. In some embodiments, the non-natural amino acid polypeptide is produced in cell lysate. In some embodiments, the non-natural anuno acid polypeptide is produced byribosomal translation.
100221 Also described herein are methods of making a polypeptide comprising a non-natural aniino acid. In soine embodiments, the methods comprise culturing cells comprising a polynucleotide or polynucleotides encoding a polypeptide, an orthogonal.RNA synthetase and/or an orthogonal tRNA
under conditions to permit expression of the polypeptide; and purifying the polypeptide from the cells and/or culture medium.
100231 Also described herein are libraries of the non-natural amino acids described herein or libraries of the non-natural amino acid polypeptides described herein, or libraries of the modified non-natural amino acid polypeptides described herein, or combination libraries thereof. Also described herein are arrays containing at least one non-natural amino acid, at least one non-natural aniino acid polypeptide, and/or at least one modified non-natural aniino acid. Also described herein are arrays containing at least one polynucleotide encoding a polypeptide comprising a selector codon. The arrays described herein are used, for exaniple, to screen for the production of the non-natural amino acid polypeptides in an organism (either by detecting transcription of the polynucleotide encoding the polypeptide or by detecting the translation of the polypeptide).
100241 Also described herein are methods for screening libraries described herein for a desired activity, or for using the arrays described herein to screen the libraries described herein, or for other libraries of compounds and/or polypeptides and/or polynucleotides for a desired activity. Also desccibed herein is the use of such activity data from library screening to develop and discover new therapeutic agents, as well as the therapeutic agents themselves.

100251 Also described herein are niethods of increasing therapeutic half life, serum half-life or circulation time of a polypeptide. In some embodiments, the methods comprise substituting at least one non-natural amino acid for any one or niore aniino acids in a naturally occurring polypeptide and/or coupling the polypeptide to a water soluble polymer.
[00261 Also described herein are methods of treating a patient in need of such treatment with an effective amount of a pharmaceutical composition which comprises a polypeptide comprising a non-natural amino acid and a pharmaceutically acceptable carrier. In some embodiments, :the non-natural amino acid is coupled to a water soluble polymer.
100271 In further or alternative embodiments are methods for treating a, disorder, condition or disease, the method comprising administering a therapeutically effective.amount of`a non-natural amino acid polypeptide comprising at least one non-natural amino acid selected from the group consisting of an indole-containing.non-natural amino acid, a carbonyl-containing non-natural amino acid, and a hydrazine-containing non-natural ainino acid. In other embodiments such non-natural amino acids have been synthetically incorporated into the polypeptide as described herein. In further or alternative embodiments such.non-natural amino acid polypeptide comprises at least one non-natural amino acid selected from amino acids of Formula I-XV, In another embodiment, such non-natural amino acid polypeptide comprises at least one natural aniino acid selected from amino acids of compounds 1-4.
100281 In further or altetnative embodiments are methods for treating, a disorder, condition or disease, the method comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one non-natural amino acid having the structure of compounds:
R H
R R3 A, ~~ N R3 R3 A' af\ N R.
3 B Rs B n R'\ R` R, RZ Rs H~O Rs ~0 2~ m~ `
Rs Ra N Rs Rs R
s ~ N Rs R3 R A~~/-~ Rs R3 R A,13wt B m Rs Rs Rj. T-r RZ Ra Rj~ N Rz H Rd 0 3 H R4.0 q Rs Rs wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker, linked at one end to an indole-containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(0)2-, -OS(O)Z-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylenc or substituted alkylene)-, -1`'(R')C(O)O-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNO-,)N(R')-, -N(R')C(NCOOR')N(R')-, -N(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')Z-N=N-, and -C(R')2-N(R')-N(R')-and each R' is independently I-I, alkyl, or substituted alkyl;
R, is H, an amino protecting.group, resin, at least one aniino acid, polypeptide, or polynucleotide; and R2 is OH, an esterprotecting group, resin, atleast one amino acid, polypeptide, or polynucleotide;
n is 0, .1, 2, or 3, and m is 0, 1, 2, or 3, provided that at least one of n or m is not 0;
wherein, each ring in structures 1, 2, 3, and 4 that has an associated R, group can contain 0, 1, or 2 R.
groups and each R. is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)L.R", where k is 1, 2, or 3, where each R" is independently H; alkyl, or substituted alkyl; or when more than one R. group is present, two R, optionally form an aryl, cycloalkyl or heterocycloalkyl;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
cach R5 is independently H, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkbxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, OH, NH2, CN, NO2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R", -C(O)ZR", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
or R3 is L-X; where, X is a selected from the group consisting of: a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a. reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation.
excitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotin.analogue; a moiety incoiporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety;
an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron densegroup; a.magnetic group; an intercalating group; a chromophore;
an energy transfer agent; a biologically active agent; a detectable label; a small molecule; an inhibitory ribonucleic acid;.a radionucleotide; a neutron-capture agent; a derivative of biotin; quantuni dot(s); a nanotransmitter; a radiotransmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecttle, a mimotope, a receptor, a reverse micelle, and any combination thereof; and L is optional, and when present.is.a linker selected from the group consisting of'alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substittited alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)0-,.-(alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(allcylene or substituted alkylene)-, -(alkylene. or substituted alkylene)=S(O)k-( alkylene or substituted alkylene)-S-, -(alkylene or substituted alkylene)-S-S-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)kN(R!)-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')z-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
when more than one RS group is present, two ortho R5 groups can optionally form a heterocycloalkyl or an aromatic heterocycloalkyl;
or the -B-itidole containing moiety together forni a bicyclic or tricyclic cycloalkyl or heterocycloalkyl coniprising at least one indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable prodrug or solvate thereof.
100291 In one embodiment, X is selected from a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group;
at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;
(00301 In one embodiment, both A and B are bonds, each R3 is H and R4 is H. In a further embodiment, each of Ri and R2 are at least one_ amino acid. In a further embodiment, each of R, and R2 are at least two amino acids. In a further embodiment, each of R, and R, are at least three.amino acids. In a further embodiment, each of R, and R2 are at least four amino acids. In a further embodiment, each of R, and R2 are at least five. amino acids: In a fttrther embodiment, each of R, and R2 are at least eix amino acids.

100311 In another embodiment are methods for treating a disorder, condition or, disease, the method comprising administering a therapeutically effective amount of a non-natuial amino acid polypeptide with a pharniaceutically acceptable carrier. In a further embodiment is a method for treating a disorder, condition or disease, wherein X is a water-soluble polymer. In another embodiment is a method for treating a disorder, condition or disease, wherein X is a derivative of polyethylene glycol. ln a further embodiment, is a method for treating a disorder, condition or disease, wherein X is a cytotoxic compound.
In a fitrther embodiment is a metliod for treating a disorder, condition or disease, wherein X is a drug.
100321 In some embodiments are methods for treating a disorder, condition or disease, wherein X is a second polypeptide. In a further embodiment are methods for treating a disorder, condition ordisease, wherein the second polypeptide is a peptide containing a non-natural amino acid polypeptide. In a further embodiment, are methods for treating a disorder, condition or disease, wherein the second polypeptide has the same amino acid structure as the non-natural amino acid polypeptide of compounds having the, structures 1-4. In another embodiment is a method for treating.a disorder; condition or disease, wherein X is a detectable label. In yet another embodiment are methods for treating a disorder, condition or disease, wherein the at least one non-natural amino acid of compounds.1-4 is incorporated at a specific site withiin the polypeptide. In.yet a further embodimejit are methods for treating a disorder, condition or disease, wherein the non-natural amino acid of compounds 1-0 is incorporated using a translation system.
(00331 In another embodiment is a method for treating a disorder, condition or disease comprising administering a therapeutically effective amount of a polypeptide comprising at least one non-natural amino acid selected from the group consisting of:
Ra k ~ NH
~ H N

R5 Ri, N R2 n R5 H R4 O Ra m Rs Ri.N Rz RS

Rs 6 Rs H Rs P-a~
ti n 1/ R5 R5 m RS R5 Rt, N R 2 ~ RS R
RS
5 Ri.N R
H RaO H RaO

wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene; substituted lower alkenylene, alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, ll heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker, linked at one end to an indole-containing moiety, the liril:er selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene; substituted lower alkenylene,.lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-,.
-O-(alkylene or substituted alkylene)-,.-S-, -S-(alkylene or substituted alkylene)-, -S(O)k-where k is 1, 2; or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)Z-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-. -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNO2)N(R')-, -N(R')C(NCOOR')N(R')-, -NT(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')a-N-N-, and -C(R')rN(R')-N(R')-and each R' is independently H, alkyl, or substituted alkyl;
R, is H, an amino protecting group, resin, at least one aniino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one aniino acid, polypeptide, or polynucleotide;
n is 0, 1, 2; or 3, and m is 0, 1, 2, or 3, providedthat at least one of n or m is not 0;
wherein, each ring in structures 1,;2, 3, and 4 that has an associated R9 group can contain 0, 1, or 2 R.
groups and each R, is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")Z, -C(O)R", -C(O)N(R")2, -OR", and -S(O)rR", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl; or when more than one R. group is present, two R, optionally form an aryl, cycloalkyl or heterocycloalkyl;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
each RS is independently H, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, OH, NH2, CN, NO2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R", -C(O)ZR", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substittited aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,. or when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
or R5 is L-X, where, X is a selected from the group consisting of: a label; a dye; a polymer=, a water-soluble polynter; a derivative of polyethylene.glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety.; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotin analogue; a nioiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar;:a redox-active agent; an amino thioacid; a toxic moiety;
an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore;
an energy transfer agent; a biologically active agent; a detectable label; a small molecule; an inhibitory ribonucleic acid; axadionucleotide; a netitron-capture agent; a derivative of biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-,-C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or. substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -(alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)-S(O)k-( alkylene:or substituted alkylene)-S-, -(alkylene or substituted alkylene)-S-S-, -S(O)kN(R')-, -N(R')C(O)N(R')-; -N(R')C(S)N(R')-,, -N(R')S(O)k-N(R')-, -N(R')-N=, -C(R')=NT-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
when more than one R5 group is present, two ortho R5 groups can optionally form a heterocycloalkyl or an aromatic heterocycloalkyl;
or the -B-indole containing moiety together form a bicyclic or tricyclic cycloalkyl or ccterocycloalkyl comprising at least one indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable prodrug or solvate thereof.
100341 In one embodiment, X is selected from a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group;
at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;
100351 In one embodiment, both A and B are bonds, each R3 is H and R4 is H. In a further embodiment, each of Ri and R2 are at least one amino acid. In a further. embodiment, each of R, and R, are at least two amino acids. In a further embodiment, each of Ri and RZ. are at least three amino acids. In a further embodiment, each of R, and R., are at least four amino acids. In a fnrther embodiment, each of R, and R2 are at least five amino acids. In a further embodiment, each of R, and R2 are at least eix amino acids.

100361 In yet another embodiment, is a niethod for treating a disorder, condition or disease further comprising administering a pharmaceutically acceptable carrier with the therapeutically effective amount of the polypeptide having the compounds of structures 5-8. In a further embodiment, is a method for treating a disorder, condition or disease, wherein Ri and R2 are both polypeptides. In yet another embodiment, is a method for treating a disorder, condition or disease, wherein X is a water-soluble polymer. In another einbodiment, is a method for treating a disorder, condition or disease, wherein X is a derivative of polyethylene glycol. In yet another embodiment, is a method.for tieating a disorder, condition or disease, wherein X is a cytotoxic compound. In yet a further embodiment,: is a method for treating a disorder, condition or disease, wherein X is a drug. In yet another embodiment, is a method for treating a disorder, condition or disease, wherein X is a second polypeptide. In yet another embodiment, is a method for treating a disorder, condition or disease, wherein the second polypeptide is a peptide containing a non-natural amino acid polypeptide.
100371 In further or alternative embodiments are methods for treating a disorder, condition or disease, the method comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one non-natural amino acid having the structure of compounds 1-4, wherein the polypeptide is a protein homologous to a therapeutic protein selected from the group consisting of: alpha- I antitrypsin, angiostatin, antihemolytic factor, antibody, antibody fragment, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, EIv'A-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte int7ammatory protein-1 alpha, monocyte inllammatory protein-i beta, RANTES, 1309, R83915, R91733, HCCI, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, coniplement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helical bundle protein, G-CSF,, glp-1, GM-CSF, glucocerebrosidase;
gonadotropin, growth factor,,growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth horinone (hGH), human serum albumin, ICAM-1, ICAM- I receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL41, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxiit B, pyrogenic exotozin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF
receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SECI, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha I, tissue plasniinogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis. factor alpha, tumor necrosis factor beta; tumor necrosis factor receptor (TNFR), urotensin, VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone receptor.

100381 In a further embodiment are methods for treating a disorder, condition, or disease, the method comprising administering compounds having the structures 5-8, wherein the at least one non-natural acid is incorporated at a specific site within the polypeptide. In a further embodiment are methods for treating a disorder, condition, or disease, the method comprising administering compounds having the structures 5-8, wherein the non-natural amino acid is incorporated using a translation system.
In a further enibodiment are methods for treating a disorder, condition, or disease, the method comprising administering compounds having the structures 5-8, wherein the non-natural amino acid is incorporated into the polypeptide using a translation system and a post translation modification system. In a further embodiment are methods for treating a disorder, condition, or disease, the method comprising administering compounds having the structures 5-8, wherein the polypeptide comprising at least one non-natural amino acid is stable for at least 1 month. In a further embodiment are methods for treating a disorder, condition, or disease, the method comprising administering compounds having the structures 5-8, wherein the polypeptide comprising at least one non-natural amino acid is stable for at least 2 weeks.
100391 In further or alternative embodiments are methods for treating a disorder, condition or disease, the 15, method comprising administering: a therapeutically effective amount of a non-natural ,amino acid polypeptide comprising at least one indole-containing non-natural amino acid and the resulting indole-containing non-natural amino acid polypeptide increases the bioavailability-of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide.
100401 In further or alternative embodiments are methods for treating a disorder, condition or disease, the method comprising administering a therapeutically effective amount of a non-natural anuno acid polypeptide comprising at least one indole-containing non-natural amino acid and the resulting indole-containing non-natural amino acid polypeptide increases the safety profile of the polypeptide relative to the homologous naturally-occurring ainino acid polypeptide.
100411 In fiu-ther or alternative embodiments are methods for treating a disorder, condition or disease, the method comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one indole-containing non-natural amino acid and the resulting indole-containing non-natural amino acid polypeptide increases the water solubility of the polypeptide relative to the homologous naturally-occurring.amino acid polypeptide.
100421 In further or alternative. embodiments are methods for treating a disorder; condition or disease, the metliod comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one indole-containing non-natural amino acid and the resulting indole-containing non-natural amino acid polypeptide increases the therapeutic half-life of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide.
100431 In further or alternative embodiments are metliods for treatntg a disorder, condition or disease,. the method comprising administering a tlierapeutically effective amount bf a non=natural amino acid polypeptide comprising at least one indole-containing non-natural amino acid and the resulting indole-containing non-natural amino acid polypeptide increases the serum half-life of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide.

100441 In further or alternative embodiments are methods for treating a disorder, condition or disease, the method comprising administering;a therapeutically effective:amount of a:non-natural amino acid polypeptide coniprising at least one indole-containing non-natural amino acid and the resulting indole-containing non-natural, amino acid polypeptide extends the circulation, time of the polypeptide relative to the homologous naturally-occurring amino.acid polypeptide.
(,0045) In further or alternative embodiments are methods for treating a disordei, condition or disease, the method comprising administering a, therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one indole-containing non-natural amino acid and the resulting, indole-containing non-iiatural ainino acid polypepride nlodulates the biological, activity of the.polypeptide relative to the homologous naturally-occurring amino acid polypeptide.
100461 In further or alternative embodiments are methods for treating a disorder, condition or disease, the method comprising administering a therapeutically effective ~amount of a-non-natural amino acid polypeptide comprising at least one indole-containing non-natural amino acid and the resulting indole-containing non-natural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide.
100471 It is to be understood that the methods and compositions described herein are not limited to the particular metliodology, protocols, cell lines, constructs, and reagents described herein and as such optionally vary. It is also to be understood that the terminology used herein is for the purpose of describing particular enibodiments only, and is not intended to limit the scope of the methods and compositions described herein, which, will be limited only by the appended claims.
100481 As used herein and in the appended claims, the singular forms "a,"
"anand "the" include plural reference unless the context clearly indicates otherwise.
100491 Unless defined otherwise, all technical and scientific xerms used herein have the same meaning as commonly understood to one of ordinary skill in the. art to which the inventions described herein belong.
Although any methods, devices, and materials similar'or equivalent to those described heiein can be used in the practice or testing of the. inventions described herein, the preferred methods, devices and materials are now described.
100501 The publications discussed herein are provided solely for theii=
disclosure prior to the filing date of the present application. Notbing herein is to be construed as an admission that the inventors described herein are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.
100511 The term "affinity label," as used heiein, refers to a label which reversibly or irreversibly binds another molecule, either to modify it, destroy it, or form a compound with it. By way of example, affinity labels include enzymes and their substrates, or antibodies and their antigens.
[0052) The terni.s "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy) refer to those alkyl groups linked to molecules via an oxygen atom, an amino group, or a sulfuuatom, respectively.
100531 The term "alkyl," by itself or as part of another molecule means, unless otherwise stated, a.straight or branched chain, or cyclic hydiocarbon radical, or: combination thereof, which optionally is fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. Cl-C1o rneans one to ten'carbons). Exainples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl; n-propyl, isopropyl, n-butyl; t-butyl, isobutyl, sec-butyl, cyclohexyl; (cyclohexyl)methyl, cyclopropylmethyl, horriologs.and isomefs of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term "alkyl," unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail herein, such as "heteroalkyl", "haloalkyl" and "homoalkyl".
100541 The term. "alkylene" by itself or as part of another molecule means a divalent radical derived from an alkane, as exemplified, by (-CHZ-),,, wherein n is 1 to about 24. By way of example only, such groups include, but are not limited to, groups having 10 or fewer carbon atoms such as the structures -CHZCHZ- and -CH2CH2CH2CI-Ii-. A "lower alkyl" or "lower alkylene" is a shorter chain.alkyl or alkylene group, generally having eight or fewer carbon atoms. The term "alkylene," unless otherwise noted, is also meant to, include those groups described hercin as "heteroalkylene."
[0055[ The terni "amino acid" refers to naturallyoccurring and non-natural amino acids, as well as amino acid analogs and aniino acid mimetics that function in a manner similar to the naturally occurring amino acids.
Naturally encoded amino acids are the 20 common amino acids (alariine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine,, phenylalaninc, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine: Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an cr-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R
group. Such analogs optionally have modified R groups (by way of example, norleucine) or optionally inodi6ed peptide backbones while still retaining the same basic chemical structure as a naturally occurring amino acid.
Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
100561 Amino acids may be. referred to herein by either their name, their three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Additionally, nucleotides, may be referred to by their commonly accepted single-letter codes.
100571 By "antibody fragment" is meant any form of an antibody other than the full-length fotni. Antibody fragments herein include antibodies that are smaller .comporients that exist within full-length antibodies, and antibodies that have been engineered. Antibody fragments include but are not limited to Fv, Fc, Fab, and (Fab')2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDRI, CDR2, CDR3, combinations of CDR's, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, and alternat.ive scaffold non-antibody molecules, bispecific antibodies, and the like (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol.
9:395-402). Another functional substructure is a single chain Fv (scFv), comprised of the variable regions of the irnmunoglobulin heavy and light chain, covalently connected by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules.
Unless specifically noted otherwise, statements and claims that use the term "antibody" or "antibodies"
specifically includes "antibody fragment" and "antibody fragnients."

100581 The term "aromatic" or "aryl", as used herein, refers to a closed ring structure which has at least one ring having a conjugated pi electron system and includes both carbocyclic aryl and heterocyclic aryl (or "heteroaryP" or "heteroaromatic") groups. The carbocyclic or heterocyclic aromatic group optionally contains from 5 to 20 ring atonu. The term includes monocyclic rings linked covalently or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups. An aromatic group can be unsubstituted or substituted. Non-limiting examples of "aroniatic" or "aryl", groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl, and phenantliracenyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described herein.
100591 For brevity, the term "aromatic" or "aryl" when used in combination with other terms (including but not limited to, aryloxy, arylthioxy, aralkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" or "alkaryl" is meant to include those radicals in which an aryl group is attached to an alkyl group (including but not liniited to, benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (including but not limited to, a methylene group) has been replaced by a heteroatom, by way of example only, by an oxygen atom. Examples of such aryl groups include, but are not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naphthyloxy)propyl, and the like.
[00601 The term "arylene", as used herein, refers to a divalent aryl radical.
Non-limiting examples of "arylene" include phenylene, pyridinylene, pyrimidinylene and thiophenylene.
Substituents for arylene groups are selected from the group of acceptable substituents described herein.
100611 The term "at least one amino acid" refers to a single amino acid, a multiplicity of aniino acids, an oligopeptide, an amino acid dimer, an aniino acid trimer, an amino acid tetramer, a polypeptide, a protein, an antibody, or any other connected chain of amino acids.
100621 A"bifunct.ional polymer", also referred to as a "bifunctional linker", refers to a polymer comprising two functional groups that are capable of reacting specifically with other moieties to form covalent or non-covalent linkages. Such moieties include, but are not limited to, the side groups on natural or non-natural amino acids or peptides which contain such natural or non-natural amino acids. By way of example only, a bifunctional linker has a functional group reactive with a group on a first peptide, and another functional group which is reactive with a group on a second peptide, whereby forming a conjugate that includes the first peptide, the bifunctional linker and the second peptide. Procedures and linker molecules for attachment of various compounds to peptides include, e.g., European Patent Application No. 188,256;
U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789. A "multi-functional polymer" also referred to as a "multi-functional linker", refers to a polymer comprising two or more functional groups that are capable of reacting with other moieties. Such moieties include, but are not limited to, the side groups on natural or non-natural aniino acids or peptides which contain such natural or non-natural amino acids. (including but not linuted to, amino acid.side groups) to form covalent or non-covalent linkages.
A bi-functional polymer or multi-functional polymer is optionally any desired length or molecular weight, and is optionally selected to provide a particular desired spacing or conformation between one or more molecules linked to a compound and molecules it binds to or the compound.
100631 'fhe term "bioavailability," as used herein, refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. Increases in bioavailability refers to increasing the rate and extent a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the gencral circulation. By way of exaniple, an increase in bioavailability is indicated as an increase in concentration of the substance or its active moiety in the blood when compared to other substances or active moieties. This method is optionally used for evaluating the bioavailability of any polypeptide.
(0064) The term "biologically active molecule", "biologically active nioiety"
or "biologically active agent"
when used herein means any substance which can affect any physical or biochemical properties of a biological system, pathway, molecule, or interaction relating to an organism, including but not limited to, viruses, bacteria, bacteriophage, transposon, prion, insects, fungi, plants, animals, and humans.
In particular, as used herein, biologically active molecules include but are not limited to any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in hunians or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles and micelles. Classes of biologically active agents that are suitable for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers,:antibiotics, fungicides, anti-viral agents, ant'r-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, microbially derived toxins, and the like.
100651 By "modulating biological activity" is meant increasing or decreasing the reactivity of a polypeptide, altering the selectivity of the polypeptide, enhancing or decreasing the substrate selectivity of the polypeptide.
Analysis of modified biological activity can be performed by comparing the biological activity of the non-natural polypeptide to that of the natural polypeptide.
100661 The tenn "biontaterial," as used herein, refers to a biologically-derived material, including but not liniited to material obtained from bioreactors and/or from recombinant methods and techniques:
100671 The term `-`biophysical probe,=" as used herein; refers to probes which can detect or monitor structural changes in molecules. Such molecules include, but are not limited to, proteins; and the "biophysical probe" is optionally used to detect or monitor interaction of proteins with other macromolecules. Exantples of biophysical probes include, but are not limited to, spin-labels, a fluorophores, and photoactivatible groups.
100681 The term "biosynthetically," as used herein, refers to any method utilizing a translation system (cellular or non-cellular), including use of at least one of the following components: a polynucleotide, a codon, a tRNA, and a ribosome. By way of example., non-natural amino acids are "biosynthetically incorporated" into non-natural amino acid polypeptides using the methods and techniques described in section VIII "ln vivo generation of polypeptides comprising non-natural amino acids".
100691 The term "biotin analogue," or also referred to as "biotin mimic", as used herein, is any molecule, other than biotin, which bind witli high affinity to avidin and/or streptavidin.
100701 The term "carbonyl" as used herein refers to a group containing at a moiety selecting from the group consisting of -C(O)-, -S(O)-, -S(O)2-, and -C(S)-, including, but not limited to, groups containing a least one ketone group, and/or at least one aldehyde groups, and/or at least one ester group, and/or at least one carboxylic acid group, and/or at least one thioestergroup. Such carbonyl groups include ketones, aldehydes, carboxylic acids, esters, and thioesters. In addition, such groups are optionally part of linear, branched, or cyclic molecules.
100711 The term "chemically cleavable group," also referred to as "chemically labile", as used herein, refers to a group which breaks or cleaves. upon exposure to acid, base, oxidizing agents, reducing agents, chemical inititiators, or radical initiators.

100721 The term "chemiluminescent group," as used herein, refers to a group which emits light as a result of a chemical reaction without the addition of heat. By way of example only, luminol (5-amino-2,3-dihydro-l,4-phthalazinedione) reacts with oxidants like hydrogen peroxide (H202) in the presence of a base and a metal catalyst to produce an excited state product (3-aminophthalate, 3-APA).
100731 The term "cliromophore," as used herein, refers to a molecule which absorbs light of visible wavelengths, UV wavelengths or IR wavelengths.
100741 The term "cofactor," as used herein, refers to an atom or molecule essential for the action of a large molecule. Cofactors include, but arenot limited to, inorganic ions, coenzymes, proteins, or some other factor necessary for the activity of enzymes. Examples include, heme in hemoglobin, magnesium in chlorophyll, and metal ions for proteins.
100751 A"con-parison wrindow," as used herein, refers a segment of any one of contiguous positions used to compare a sequence to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Such contiguous positions include, but are pot limited to a group consisting of from about to about 600 sequential units, including about 50 to about 200 sequential tinits, and about 100 to about 150 15 sequential units. By way of example only, such sequences include polypeptides and polypeptides containing non-natural amino acids, with the sequential units include, but are not limited to natural and non-natural amino acids. In addition, by way of example only, such sequences include polynucleotides with nucleotides being the corresponding sequential units. Methods of alignment of sequences for comparison include, but are not liniited to, the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.
2:482c, by the homology 20 alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).
100761 By way of example; an algorithm which is used to determine percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410;
respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The BLAST
algorithm parameters W; T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=4 and a.
comparison of both strands. For aniino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E)' of 10, and the BLOSUM62 scoring matrix (see Henikoffand Henikoff (1992) Proc. Natl.
Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm is typically performed with the "low complexity"
filter turned off.
100771 The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Nati. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.

100781 The term "conservatively modified variants" applies to both natural and non-natural amino acid and natural and non-natural nucleic acid sequences, and combinations thereof. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those natural and non-natural nucleic acids which encode identical or essentially identical natural and non-natural amino acid sequences, or where the natural and non-natural nucleic acid does not encode a natural and non-natural amino acid sequence, to essentially identical sequences. By way of example, becauseof the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the aniino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Thus by way of example every natural or non-natural nucleic acid sequence herein which encodes a natural or non-natural polypeptide also describes every possible silent.variation of the natural or non-natural nucleic acid. Each codon in a natural or non-natural. nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical tnolecule. Accordingly, each silent variation of a.natural and non-natural nucleic acid.which encodes a natural and non-natural polypeptide is implicit in each described sequence.
100791 As to amino acid sequences,, individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single natttral and non-natural amino acid or a small percentage of natural and non-natural amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the deletion of an anvno acid, addition of an amino acid, or substitution of a natural and non-natural amino acid with a chemically similar.aniino acid.
Conservative substitution tables, available in the scientific literature, provide funetionally sinular natural amino acids. Such conservatively modified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the methods and compositions described herein.
100801 The following eight groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) .Isoleucine (I)õLeucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine:(T); and 8) Cysteine (C), Methioriine (M) (see, e.g., Creighton, Proteins:Structures and Molecular Properties (W H
Freeman & Co.; 2nd edition (December 1993).
100811 The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively. Thus, a cycloalkyl or heterocycloalkyl include saturated, partially unsaturated and fully unsaturated ring linkages. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. The heteroatom includes, but is not limited to, oxygen, nitrogen or sulfur. Examples of cycloalkyl include,:but are not limited to, cyclopcntyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycioheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5;6-tetrahydropyridyl),.1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-y1, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,. 2-piperazinyl, and the like: Additionally, the term encompasses. multicyclic structures, including but not limited to;
liicy.clicand tricyolic ring structures. Similarly, the terni "heterocycloalkylene" by itself or, as, part of-another molecule means a divalent radical derived from heterocycloalkyl,and the term "cycloalkylene" by itself or as part.of another niolecule means a divalent radical derived from cycloalkyl.
100821 The term "cyclodextrin," as used herein, refers to-cyclic caibohydrates consisting of at least six to eight glucose molecules in a ring formation. The outer part of the ring contains water soluble groups; at the center of the ring:is a relatively nonpolar cavity able to accommodate small molecules.
100831 The term "cytotoxic," as used herein, refers to a compound which harms cells.
100841 The term "desired functionality," as used herein refers to any one of the following goups: a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol;
a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibod.y or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a bioniaterial; a nanoparticle; a spin ,1abe1; a fluorophore; a metal-containing moiety;
a radioactive moiety; a novel functional group; a group that covaleiitly or noncovalently interacts with other molecules; a pliotocaged moiety; an actinic, radiation excitable moiety; a ligand; ;a photoisomerizable nwiety;
biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent;, an amino thioacid; a toxic moiety;
an isotopically labeled moiety; a biophysical probe; a. phosphorescent group;
a chemiluminescent group; an electron dense gro.up; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent (in which case, the biologically active agent can.include an agent with therapeutic activity and the non-natural amino acid polypeptide or modified non-natural anlino acid can serve either as a co-therapeutic agent with the attached therapeutic agent or as a means for delivery the therapeutic agent to a desired site within an organism); a detectable label; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron-capture agent; a derivative of biotin; quantum dot(s); a nanotransntitter; a radiotransnritter; an abzyme, an activated coniplex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a iguide R.NA, a saponin, a shuttle vector, a niacromolecule, a mimotope, a receptor, a reverse micelle, and any combination thereof.
100851 The term "hydrazine,"as used herein, refers to groups/molecules comprising at least one hydrazine functional group.
100861 The term "detectable label," as used herein, refers to a label. which is optionally observable using analytical, techniques including, but not limited. to, fluorescence, chemiluminescence,._electron-spin resonance;
ultraviolet/visible absorbance spectroscopy, mass spectrome`try, nuclear magnetic resonance, magnetic resonance,:and electxochemical methods.

100871 The term "carbonyl" as used herein refers to a groups/molecules containing at least one aldehyde or one ketone.
100881 7'he term "drug," as used herein, refers to any substance used in the prevention, diagnosis, alleviation, treatment, or cure of a disease or condition.
[00891 The term "dye," as used herein, refers to a soluble, coloring substance which contains a chromophore.
100901 The term "effective amount," as used herein, refers to a sufficient amount of an agent or a compound being adininistered which will relieve: to some extent one or more of the symptonis of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, `symptoms, or causes of a disease, or any other desired alteration of a biological system. By way of example,. an agent. or a cornpound being administered includes, but' is not limited to, a natural amino acid polypeptide, non-natural amino acid polypeptide, niodified natural amino acid polypeptide, or modified non-amino acid polypeptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be administered for prophylactic, enhancing, and/or therapeutic treatments. An appropriate "effective" amount in any individual case is optionally detemtined using techniques, such as a dose escalation study.
100911 The term "electron dense group," as used herein, refers to a group which scatters electrons when irradiated with an electron beam. Such groups include, but are not limited to, ainmoniuni molybdate, bismuth subnitrate cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, hexamethylene tetramine, 98.5%, indiuni trichloride anhydrous, lanthanum nitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, phosphotungstic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver proteinate (Ag Assay: 8.0-8.5%) "Strong", silver tetraphenylporphin (S-TPPS), sodium chloroaurate, sodium tungstate, thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate, and vanadyl sulfate.
[00921 The term "energyiransfer agent," as used herein, refers to ainolecule which can either donate or accept energy from another molecule. By way of example only, fluorescence resonance energy transfer (FRET) is a dipole-dipole coupling process by which the excited-state energy of a:
fluoreseence donor molecule is non-radiatively tratisferred to an unexcited.acceptor molecule which then tluorescently enrits the donated energy at a longer wavelength.
100931 The terms "enhance" or "enhancing" means to increase or prolong either in potency or duration a desired effect. By way of example, "enhancing" the effect of therapeutic agents refers to the ability to increase or prolong, either in potency or duration, the effect of therapeutic agents on during treatment of a disease, disorder or condition. An "enhancing-effective amount," as used herein, refers to an amount adequate to enhance the effect of a therapeutic agent in the treatment of a disease, disorder or condition. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.
100941 As used herein, the term "eukaryote" refers to organisms belonging to the :phylogenetic domain Eucarya, including but not limited to animals (including but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to, monocots, dicots, and algae), fungi, yeasts, flagellates, microsporidia, and protists.

100951 The term "fatty acid," as used herein, refers to carboxylic acids with about C6 or longer hydrocarbon side chain.
100961 The term "fluoropliore," as used herein, refers to a molecule which upon excitation emits photons and is thereby fluorescent.
100971 The terms "functional group", "active moiety", "activating group", "leaving group", "reactive site", "chenucally reactive group" and "chemically reactive moiety," as used herein, refer to portions or units of a molecule at which chemical reactions occur. The terms are somewhat synonymous in the chenucal arts and are used herein to indicate the portions of molecules that perform some function or activity and are reactive with other molecules.
[0098] The term "halogen" includes fluorine, chlorine, iodine, and broniine.
100991 The term "haloacyl," as used herein, refers to acyl groups which contain halogen moieties, including, but not limited to, -C(O)CH3, -G(O)CF3i -C(O)CHZOCH3, and the like.
.1001001 The term."haloalkyl," as used herein,.refers to alkyl groups which contain halogen moieties, including, but not linuted to, -CF3 and -CH2CF3 and the like.
[001011 The term "heteroalkyl," as used herein, refers to, straight or branched chain, or cyclic hydrocarbon radicals, or combinations thereof, consisting of an alkyl group and at least one. heteroatom. selected from the group consisting of 0, N, Si and S, and wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroitom(s) 0, N and S
and Si are optionally placed at any interior position of the heteroalkyl group or at the position. at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CHZ-CHZ-O-CH3, -CH2-CH2-NH-CH3, -CHZ-CH2-N(CH,3)-CH3, -CH2-S-CHZ-CH3, -CH2-CHZ,-S(O)-CH3, -CH2_-CH2-S(0)2-CH3i -CH=CH-O-CH3, -Si(CH3)3, -CHZ-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. In addition, up to two heteroatoms are optionally consecutive, such as, by way of example, -CH2-NH-OCH3 and -CHZ-O-Si(CH3)3.
1001021 The terms "heterocyclic-based linkage" or "heterocycle linkage" refers to a moiety formed from the reaction of a carbonyl group with a hydrazine group. The resulting reaction product is a heterocycle, including a heteroaryl group or a heterocycloalkyl group. The resulting heterocycle group serves as a chemical link between a non-natural amino acid or non-natural amino acid polypeptide and another fiuictional group. In one einbodiment, the heterocycle linkage includes a nitrogen-containing heterocycle linkage, including by way of example only a a pyrrole linkage, 'an indole linkage, a benzodiazepine linkage, and a pyrazalone linkage.
1001031 Similarly, the term ' heteroalkylene" Tefers to. a divalent radical derived from heteroalkyl, as exemplified, but not linvted by, -CH2-CH2-S-CHZ-CHZ- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the same or different heteroatoms can also occupy either or both of the chain termini (including but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking -group is written. By way of example, the formula -C(0)2R'-represents both -C(0)2R'- and -R'C(O)z-.
1001041 The term "heteroaryl" or "heteroaromatic," as used herein, refers to aryl groups which contain at least one heteroatom selected from N, 0, and S; wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quatemized. Heteroaryl groups are optionally substituted or unsubstituted. A
heteroaryl group is optionally attached to the remainder of the molecule through a heteroatom. Non-limiting exaniples of heteroaryl groups include I-pyrrolyl, 2-pyrrolyl, -3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyriniidyl, 5-benzothiazolyl, purinyl, 2-benzinvdazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
1001051 The term "homoalkyl," as used herein refers to alkyl groups which are hydrocarbon groups.
(00106( The term "identical," as used herein, refers to two or more sequences or subsequences which are the same. In addition, the term "substantially identical," as used herein, refers to two or more sequences which have a percentage of sequential units which are the same when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using comparison algorithms or by manual alignnient and visual inspection. By way of example only, two or more sequences are "substantially identical" if the sequential units are about 60% identical, about 65% identical, about 70%
identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95%
identical over a specified region.
Such percentages to describe the "percent identity" of two or more sequences.
The identity of a sequence can exist over a region that is at least about 75-100 sequential units in length, over a region that is about 50 sequential units in length, or, where not specified, across the entire sequence. This definition also refers to the complement of a test sequence. By way of example only, two or more polypeptide "sequences are identical when the amino acid residues are the same, while two or more polypeptide sequences are "substantially identical" if the amino acid residues are about 60% identical, about 65 /n identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95%
identical over a specified region.
The identity can exist over a region. that is at least about 75-100 amino acids in length, over a region that is about 50 anvno acids in length, or, where not specified, across the entire sequence of a polypeptide sequence. In addition, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are the same, while two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70%
identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95%
identical over a specified region. The identity can exist over a region that is at least about 75-100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, where not specified, across the entire sequence of a polynucleotide sequence.
1001071 For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or altemative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
1001081 The terni "immunogenicity," as used herein, refers to an antibody response to administration of a therapeutic drug. The immunogenicity toward therapeutic non-natural amino acid polypeptides can be obtained using quantitative and-qualitative assays for detection of anti-non-natural amino acid polypeptides antibodies in biological fluids. Such assays include, but are not litnited to, Radioinimunoassay (RIA), Enzyme-linked immunosorbent assay ( ELISA), luminescent immunoassay (LIA), and fluorescent inununoassay (FIA).
Analysis of immunogenicity toward therapeutic non-natural amino acid polypeptides involves comparing the antibody response upon administration of therapeutic non-natural amino acid polypeptides to the antibody response upon administration of therapeutic natural amino acid polypeptides.

1001091 The term "intercalating agent," also referred to as "intercalating group," as used herein, refers to a chemical that can insert into the intramolecular space of a molecule or the intermolecular space between molecules. By way of example only an intercalating agent or:group.is a molecule which inserts into the stacked bases of the DNA double helix.
1001101 The term "isolated," as used herein, refers to separating, and removing a component of interest from components not of interest. Isolated substances can be in either a dry or semi-dry state, or in solution, including but not liniited to an aqueous solution. The isolated component can be in a homogeneous state or the isolated component can be a part of a pharmaceutical composition that comprises additional pharmaceutically acceptable carriers and/or excipients: Purity and homogeneity are detennined using analytical chemistry techniques including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography. In addition, when a component of interest is isolated and is the predominant species present in a preparation, the component is described herein as substantially purified. The term "purified,"
as used herein, refers to a component of interest which is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or greater pure. By way of example only, nucleic acids or proteins are "isolated" when such nucleic acids or proteins are free of at Ieast some of the cellular components with which it is associated in the natural state, or that the nucleic acid or protein has been concentrated to a level greater than the concentration of its in vivo or in vitro production. Also, by way of exaniple, a gene is isolated when separated from open reading frames which flank the gene and encode a protein other than the gene of interest.
1001111 The term "label," as used.herein, refers to asubstance which is incorporated into a conipound and is readily detected, whereby its physical distribution is optionally detected and/or monitored.
1001121 The term "linkage;" as used herein to refer to.bonds or chemical moiety formed from a chemical reaction between the functional group of a linker and anothermolecule. Such bonds include, but are_not limited to, covalent linkages aiid non-covalent bonds, while such chemical moieties include, but are not limited to, esters, carbonates, imines phospliate esters, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide.linkages. Hydrolytically stable linkages means that the linkages are-substantially stable in water and do not react with water at useful pH values, including but not limited to;
under physiological conditions for an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradable linkages nleans that the linkages are degradable in water or in aqueous solutions, including for example, blood. Enzyrnatically unstable or degradable linkages means that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the temiinal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG
carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent.
Other hydrolytically degradable linkages include but are not limited to carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol;
orthoester linkages that are the reaction productof a formate and an alcohol; peptide linkages formed by an amine group, including but not.limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polytner, and a 5' hydroxyl group of an 6ligonucleo6de.
1001131 The terms "medium" or "media," as used herein, refer to any culture medium used to grow and harvest cells and/or products expressed and/or secreted by such cells. Such "medium"
or "media" include, but are not limited to, solution, solid, semi-solid, or rigid supports that support or contain any host cell, including, by way of example, bacterial host cells,~yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host.cells, E. coli, or Pseudomonas host cells, and cell.contents.
Such "medium' ' or. "inedia" includes,, but is,not limited to,. medium or.
media in which the host cell has been grown into which a polypeptide has been secreted, including medium either before or'after a.proliferation step.
Such "medium" or "media" also includes; but is not limited to, buffers or reagents'that contain host cell.lysates, by way of "example a polypeptide produced intraeel[ularly-and the host cells are lysed or disrupted to release the polypeptide.
[001141 The terni "metabolite," as used herein, refers to a derivative of acompound, by way of example natural aniino acid polypeptide, 'a non-natural amino acid polypeptide, a modified'natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, that is formed when the compound, by way of example natural amino acid polypeptide, non-natural amino acid polypeptide, modified natural aniino acid polypeptide, or modified non-natural amino acid polypeptide, is metabolized. The term "pharmaceutically active metabolite" or "active metabolite" refers to a biologically active derivative of a compound, by -way of example natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, that is formed when such a compound, by way of example a natural amino acid polypeptide, non-natural amino acid polypeptide, niodified natural amino acid polypeptide, or modified non-natural anlino acid polypeptide, is nietabolized.
1001.151 The terni "metabolized," as used herein, refers to the sum of the processcs by which a particular substance is changed by an organism. Such processes include, but are not limited to, hydrolysis reactions and reactions catalyzed by enzymes. Further information on metabolism is obtained from The Pharmacological Basis of:Therapeutics, 9th Edition, McGraw-Hill (1996). By way of example only, metabolites of natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides, are identified either by administration of the natural anuno acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid.polypeptides to a host and analysis of tissue- samples from the host, or by incubation of natural artmino acid polypeptides, non-natural amino acid polypeptides, modified natural anuno acid polypeptides, or modified non-natural amino acid polypeptides with hepatic cells in vitro and analysis of the resulting compounds.
1001161 The tenn "metal chelator," as used herein, refers to a molecule which fornrnis a metal complex with metal ions. By way of example, such molecules form two or more coordination bonds with a central inetal ion and form ring structures.
100117] The term "metal-containing moiety," as used herein, refers to a group which contains a metal ion, atom or particle. Such moieties include, but.are not limited to, cisplatin, chelated metals ions (such as nickel, iron, and platinuni), and metal nanoparticles (such as nickel, iron, and platinum).

1001181 The term "moiety incorporating a heavy atom,!' as used herein, refers to a group which incorporates an ion. of atom which is usually heavier than carbon. Such ions or atoms include, but are not, timited to, silicon, tungsten, gold, lead, and uranium.
(001191 The term "modified," as used herein refers to the presence of a change to a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide or a non-natural amino acid polypeptide. Such changes, or modifications, are optionally obtained by posYsynthesis modifications of natural amino acids, non-natural amino acids, natural amino acid polypeptides or non-natural amino acid polypeptides, or by co-translational, or by post-translational modification of natural anuno acids, non-natural amino acids, natural amino acid polypeptides or non-natural amino acid polypeptides. The -form "modified or unmodified"
means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide being discussed are optionally modified, that is, he natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide under discussion can be modified or unmodified.
1001201 As used herein, the tcrm.`modulated serum half-life" refers to positive or negative changes in the circulating half-life of a modified biologically active molecule relative: to its non-modified form. By way of example, the iriodified biologically active, molecules include, ,but are not limited to,.natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide. By way ofexample, serum half-life, is measured by, taking blood samples at, various time points after administration of the biologically active-molecule or modified biologically active molecule, and deternvning<the concentration of that molecule in each.sampie. Correlation,of the. serum concentration with time allows calculation of the serum half-life. By way of example, modulated serum half=.life is an increased in serum half-life, which enables an imp,roved dosing regimens or avoid toxic effects. Such increases in serum are at:least about two fold, at least about three-fold, at least about five-fold, or at least about ten-fold. This method is optionally used for evaluating the serum half-life of any polypeptide.
1001211 The term "modulated therapeutic half-life," as used herein, refers to positive or negative change in the half-life of-the therapeutically effective amount of a modified biologically active molecule, relative to its non-modified form. By way of exaniple, the modified biologically active molecules include, but are not linuted to, natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-naturat amino acid polypeptide. By way of example, therapeutic half-life is, measured by measuring pharmacokinetic and/or pharmacodynamic properties of the, molecule at various time points after administration. Increased therapeutic half-life optionally enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoids an undesired effect. By way of example, the increased therapeutic half-life results from increased potency, increased or decreased binding of,,the modified niolecule to its target, an increase or decrease in another paranieter,or mechanism of action of the, non-modified molecule, or an increased or decreased breakdown of the molecules by enzymes such as, by way of example .only, proteases. This method is used for evaluating the therapeotic ha1F life.of any polypeptide.
1001221 The term "nanoparticle;" as used herein, refers to a particle which has a particle size between about 500 nm'to about I nm.
1001231 The term "near-stoichiometric," as used herein, refers to the ratio of the moles of compounds participating in a chemical reaction being about 0.75 to about 1.5.

1001241 As used herein, the term "non-eukaryote" refers to non-eukaryotic organisms. By way of example, a non-eukaryotic organism belongs to the Eubacteria, (which includes but is not limited to, Escherichia coli, Thertnus thermophilus, or Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida), phylogenetic domain, or the Archaea, which includes, but is not lirnited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, or Halobacterium such as Haloferax volcanii and Halobacterium species NRC-l, or phylogenetic doniain.
1001251 A, "non-natural aniino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrolysitte or selenocysteine. Other synonymous terms are "non-naturally encoded amino acid," "unnatural amino acid," "non-naturally-occurring amino acid," and variously hyphenated and non-hyphenated versions thereof. The term "non-natural amino acid" includes, but is not limited to, amino acids which occur naturally by modifcation of a naturally encoded amino acid (including but not lirnited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into a growing polypeptide chain by the translation complex. Exaniples of naturally-occurring amino acids that are not naturally-encoded include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and 0-phosphotyrosine.
Additionally, the term "non-natural amino acid" includes, but is not limited to, amino acids which do not occur naturally and are obtained synthetically or are obtained by modification of non-natural amino acids.
1001261 'llte term "nucleic acid," as used herein, refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in either single- or double-stranded form. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogues of natural nucleotides which have similar binding properties as a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides; (ii) oligonucleotide analogs including; but are not limited to, PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like); (iii) conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences and sequence explicitly indicated. By way of example, degenerate codon substitutions are achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
1001271 The term "oxidizing agent," as used herein, refers to a compound or material which removes an electron from a compound being oxidized. By way of example oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystanvne, oxidized dithiothreitol, oxidized erythreitol, and oxygen. Axvide variety of oxidizing agents are suitable for use in the methods and compositions described herein.
1001281 The term "pharmaceutically acceptable", as used hcrein, refers to a material, including but not limited, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material is administered to an individual without causing undesirable biological effects or interacting in a deleterious nianner with any of the components of thecomposition: in which it is contained.
1001291 The term "photoaffinity label," as used herein, refers to a label with a- group, which, upon exposure to light, forms a linkage with a molecule for which the label has an affinity: By way of example only, such a linkage is covalent or non-covalent.

1001301 The term "photocaged moiety," as used herein, refers to a group which, upon illumination at certain wavelengths, covalently or non-covalently binds other ions or molecules.
1001311 The term "photocleavable group," as used herein, refers to a group which breaks upon exposure to light.
1001321 The term "photocrosslinker," as used herein, refers to a conipound comprising two or more functional groups which, upon exposure to light, are reactive and form a covalent or non-covalent linkage with two or more monomeric or polymeric molecules.
1001331 The term "photoisomerizable moiety," as used herein, refers to a group wherein upon illumination with light changes from one isomeric form to another.
1001341 The term "polyalkylene glycol," as used herein, refers to linear or branched polymeric polyether polyols. Such, polyall.ylene glycols, including, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed, for exatnple, in commercial supplier catalogs, such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). By way of example only, such polymeric polyether polyols have average molecular weights within a desired polymer molecular weight range.
1001351 The term "within a desired polymer molecular weight range," as used herein means between about 0.1 kDa to about 100 kDa. By way of example, between about 100 Da and about 100,000 Da or more. The niolecular weight of the polymer is between, for example, about 100 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da.
In some enibodiments, the molecular weigllt of the polymer is between about 100 Da and about 40,000 Da.
In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da.
In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da.
In sonie embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the polymer niolecule is a branched polymer. The molecular weight of the branched chain polymer is between, for example, about 1,000 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of the branched chain polymer is between about 1,000 Da and about 50,000 Da.
In some embodiments, the molecular weight of the branched chain polymer is between about 1,000 Da and about 40,000 Da. In some enibodiments, the molecular weight of the branched chain polymer is between about 5,000 Da and about 40,000 Da. In sonie embodiments, the molecular weight of the branched chain polymer is between about 5,000 Da and about 20,000 Da.
1001361 The term "polymer," as used herein, refers to a molecule composed of repeated subunits. Such niolecules include, but are not limited to, polypeptides, polynucleotides, or polysaccharides or polyalkylene glycols.

100137] The terms''polypeptide;"'`peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-natural amino acid.
Additionally, such "polypeptides; " "peptides" and "proteins" include aniino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
1001381 The term "post-translationally modified" refers to any modification of a natural or non-natural amino acid which occurs after such an amino acid has been translationally incorporated into a polypeptide chain. Such inodifications include, but are not limited to, co-translational in vivo modifications, co-translational in vitro modifications (such as in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro niodifications.
1001391 The terms "prodrug" or "pharmaceutically acceptable prodrug," as used herein, refers to an agent that is converted into the parent drug in vivo or in vitro, wherein which does not abrogate the biological activity or properties of the drug, and is relatively nontoxic, i.e., the material is administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Prodrugs are generally drug precursors that, following administration to a subject and subsequent absorption, are converted to an active, or a more active species via some process, such as conversion by a metabolic pathway. Some prodrugs have a chemical group present on the prodrug that renders it less active and/or confers solubility or some other property to the drug. Once the chemical group has been cleaved and/or modified from the prodrug the active. drug is generated.
Prodrugs are converted into active drug within the body through enzymatic or non-enzymatic reactions. Prodrugs, for example, provide iniproved physiochemical properties such as better solubility, enhanced delivery characteristics, such as specifically targeting a particular cell, tissue, organ or ligand, and iniproved therapeutic value of the drug. The benefits of such prodrugs include, but are not limited to, (i) ease of administration compared with the parent drug; (ii) the prodrug is bioavailable by oral administration whereas the parent is not; and (iii) the prodrug has improved solubility in pharmaceutical compositions compared with the parent drug. A pro-drug includes a pharmacologically inactive, or reduced-activity, derivative of an active diug.
Prodrugs are designed, for example, to modulate the amount of a drug or biologically active molecule that reaches a desired site of action through the manipulation of the properties of a drug, such as physiochemical, biopharmaceutical, or pharmacokinetic properties. An example, without limitation, of a prodrug would be a non-natural amino acid polypeptide which is administered as an ester (the "prodrug") to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water-solubility is beneficial. Prodrugs are also designed, for example, as reversible drug derivatives, for use as modifiers to enhance drug transport to site-specific tissues.
1001401 The term "prophylactically effective amount," as used herein, refers that amount of a composition containing at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide prophylactically applied to a patient which will relieve to some extent one or more of the symptoms of a disease, condition or disorder being treated. In such prophylactic applications, such amounts depend, for example, on the patient's state of health, weight, and the like.

1001411 The term "protected," as used lierein, refers to the presence of a "protecting group" or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or a hydrazide, the protecting group is selected from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the chemically reactive group is a thiol,,the protecting group is orthopyridyldisulfide; and (iii) if the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group, the-protecting group is benzyl or an.alkyl group such as methyl, ethyl, or tert-butyl.
1001421 By way of example only,=blocldng/protecting groups are selected from:
Hz H2 0 H C~CC~H~ C~O H2C C~HO` H3C
2 H2 2 ~O( allyl Bn Cbz alloc Me H2 H3C\ CH3 0 H3C' C-_ (H3C)3C-~ (H3C)3C- SI-I (H3C)3&\~ O

Et t-butyl TBDMS Teoc H2 ~
C~ O HzC-O
(CH3)IC'0~ / I (C8H513C'_ H3C~
O. H3C0 ~ ~
BOC pMBn trityl acetyl Fmoc 1001431 Additionally, protecting groups include, but are not liniited to, including photolabile groups such as Nvoc and MeNvoc and other protecting groups, such as those described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY,.1999.
1001441 The term "radioactive moiety," as used herein, refers to a group whose nuclei spontaneously give off nuclear radiation, such as alpha, beta, or gamma particles; wherein, alpha particles are helium nuclei, beta particles are electrons, and gamma particles are high energy photons.
1001451 The term "reactive compound," as used herein, refers to a compound which under appropriate conditions is reactive toward another atom, molecule or compound.
1001461 The term "recombinant host cell," also referred to as "host cell,"
refers to a cell which includes an exogenous polynucleotide, wherein the methods used to insert the exogenous polynucleotide into a cell include, but are not limited to, direct uptake, transduction, orf-mating, to create reconibinant host cells. By way of exaniple only, such exogenous polynucleotide is a nonintegrated vector, including but not limited to a plasmid, or is integrated into the host genome.
1001471 'rhe term "redox-active agent," as used herein, refers to a molecule which oxidizes or reduces another molecule, whereby the redox active agent becomes reduced or oxidized. Examples of redox active agent include, but are not limited to, ferrocene, quinones, Ru2'13+ complexes, Co2'/3+ complexes, and Os2"3' complexes.

1001481 The tenn "reducing agent," as used herein, refers to a compound or material which is capable of adding an electron to a compound being reduced. By way of example reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. Such reducing agents are used, by way of example only, to niaintain sulfhydryl groups in the reduced state and to reduce intra- or intermolecular disulfide bonds.
1001491 The term "resin," as used herein, refers to high molecular weight, insoluble polymer beads. By way of example only, such beads are used as supports for solid phase peptide synthesis,, or sites for attachment of molecules prior to purification.
[001501 The term "saccharide," as used herein,, refers to a series of carbohydrates including but not limited to sugars, monosaccharides, oligosaccharides, and polysaccharides.
1001511 The term "safety" or "safety profile;" as used herein, refers to side effects that are related to administration of a drug relative to the number of times the drug has been adrriinistered. By way of example, a drug which has been administered many times and produced only mild or no side effects is said to have an excellent safety profile. This method is used, for example, for evaluating the safety profile of any polypeptide.
1001521 The term "spin label," as used herein, refers to molecules which contain an atom or a group of atoms exhibiting an unpaired electron spin (i.e. a stable paramagnetic group) that can be detected by electron spin resonance spectroscopy and can be attached to another molecule. Such spin-label molecules include, but are not liniited to, nitryl radicals and nitroxides, and are single spin-labels or double spin-labels.
1001531 The term "stoichiometric," as used herein, refers to the ratio of the moles of compounds participating in a chemical reaction being about 0.9 to about I.I.
1001541 The tenn "stoichionietric-like," as used herein, refers to a cheniical reaction which becomes stoichiometric or near-stoichiometric upon changes in reaction conditions or in the presence of additives. Such changes in reaction conditions include, but are not limited to, an increase in temperature or change in pH. Such additives include, but are not limited to, accelerants.
1001551 The term "subject" as used herein, refers"to an animal which is the object of treatment, observation or experiment. By way of example only, a subject is, but is not limited to, a mammal including, but not limited to, a human.
1001561 The term "substantially purified," as used hereinõrefers to a component of interest that is substantially or essentially free of other components which normally accompany or interact with the component of interest prior to purification. By way of example only, a component of interest is "substantially purified" when the preparation of the coniponent of interest contains less than about.30%; less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a "substantially purified" component of interest has a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater. By way of example only, a natural amino acid polypeptide or a non-natural amino acid polypeptide is purified from a native cell, or host cell in the case of recombinantly produced natural amino acid polypeptides or non-natural amino acid polypeptides. By way of example a preparation of a natural anuno acid polypeptide or a non-natural amino acid polypeptide is "substantially purified" when the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contanunating material. By way of example when a natural amino acid polypeptide or a non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide or non-natural amino acid polypeptide is present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. By way of example when a natural amino acid polypeptide or a non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide or non-natural amino acid polypeptide is present in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about lg/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about I Omg/L, or about I mg/L
or less of the dry weight of the cells. By way of example, "substantially purified" natural aniino acid polypeptides or non-natural amino acid polypeptides has a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about'99% or greater as deterniined by appropriate methods, including, but not limited to, SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.
1001571 The tetm "substituents" also referred to as "non-interfering substituents" "refers to groups which are used to replace another group on a molecule. Such groups include, but are not limited to, halo, Cl-Clo alkyl, C2-CQ alkenyl, C2-Clo alkynyl, C-Cio alkoxy, C5-C12 aralkyl, C3-C12 cycloalkyl, C4-Cõ cycloalkenyl, phenyl, substituted phenyl, toluolyl, xylenyl, biphenyl, C2-C12 alkoxyalkyl, CS-Cõ
alkoxyaryl, CS-.Ci2 aryloxyalkyl, C7-C12 oxyaryl, CI-C6 alkylsulfinyl, Cl-Clo alkylsulfonyl, -(CHZ),n O-(Cj-Co alkyl) wherein m is.from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -NO2, -CN, -NRC(O)-(Ci-C,o alkyl), -C(O)-(CI-COalkyl), C2-C10 alkthioalkyl, -C(O)0-(Ci-Clo alkyl), -OH, -SO2, =S, -COOH, -NR2, carbonyl, -C(O)-(CI-Cio alkyl)-CF3, -C(O)-CF3, -C(O)NR2, -(CI-Clo aryl)-S-(C6-CIO
aryl), -C(O)-(C6-Cio aryl), -(CH,),,; O-(CH2),,; O-(Cj-Cio alkyl) wherein each m is from 1 to 8, -C(O)NR2, -C(S)NR2, -SOZNR2, -NRC(O)NR2, -NRC(S)NR2, salts thereof, and the like. Each R
group in the preceding list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl. Where substituent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left; for example, -CH2O- is equivalent to -OCH2-.
(001581 By way of example only, substituents for alkyl and heteroalkyl radicals (including those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkcnyl, and heterocycloalkenyl) includes, but is not limited to: -OR, =0, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -COZR, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR,, -NR(O)zR, -NR-C(NRz)=NR, -S(O)R, -S(O)2R, -S(O)_NR,, -NRSO2R, -CN and -NO2. Each R group in the preceding list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1 or 2 halogens, substituted or unsubstituted.alkyl, alkoxy or thioalkoxy groups, or aralkyl groups. When two R groups are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is meant to include, but not be liniited to, i-pyrrolidinyl and 4-morpholinyl.
1001591 By way of example, substituents for aryl and heteroaryl groups include, but are not linzited to, -OR, =0, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -C02R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NRZ, -NR(O),R, -NR-C(NR2)=NR, -S(O)R, -S(0)2R, =S(O)ZNRZ, -NRSOZR, -CN, -NO2, -R, -N3, -CH(Ph)2, iluoro(Ci-C4)alkoxy, and fluoro(Ci-C4)alkyl, in a number ranging from zero to the total number of open valences on the aroniatic ring system; and where each R group in the preceding list includes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl and heteroaryl.
1001601 The term "therapeutic protein," as used herein, refers to any one or all of the following polypeptides/proteins: alpha-I antitrypsin, angiostatin, antihemolytic.
factor, antibody, antibody fragment, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial pep6de, C-X-C
chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b,.gTo-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF), complement facto'r 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-l, epidermal growth factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-ll, interferon (IFN), IFN-alpha, IFN-beta, IFN-ganuna, any interferon-like molecule or meniber of the IFN
fanuly, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, 1L-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene pcoduct, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotozin C, pyy, relaxin; renin, SCF, small biosynthetic. protein, soluble complement receptor 1, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SECI, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF), tunior necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor ('ITIFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.
1001611 The term "therapeutically effective amount," as used herein, refers to the amount of a composition containing at least one non-natural anuno acid polypeptide and/or at least one modified non-natural amino acid polypeptide administered to a patient already suffering from a disease, condition or disorder, sufficient to cure or at least partially arrest, or relieve to some extent one or nlore of the symptonis of the disease, disorder or condition being treated. The effectiveness of such compositions depend conditions including, but not limited to, the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. By way of example only, therapeutically effective amounts are determined by methods, including but not limited to a dose escalation clinical trial.

[001621 The term "thioalkoxy," as used herein, refers to sulfur containing alkyl groups linked to molecules via an oxygen atom.
1001631 7'he term "thermal melting point" or Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of probes complementary to a target hybridize to the target sequence at equilibrium.
1001641 The term "toxic moiety," as used herein, refers to a compound which can cause harm or death.
1001651 The terms "treat," "treating" or "treatment", as used herein, include alleviating, abating or ameliorating a disease or condition symptonis, preventing additional symptoms,, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease 1.0 or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by'the disease or condition, or stopping the symptoms of the disease or condition. The terms "treat," "treating" or "treatment", include, but are not limited to, prophylactic and/or therapeutic treatments.
[001661 As used herein, the term "water soluble polymer" refers to any polymer that is soluble.in aqueous solvents. Such water soluble polymers include, but are not limited to, polyethylene glycol, polyethylene. glycol propionaldehyde, mono Cl-Cln alkoxy or aryloxy derivatives thereof (described in U.S. Patent No. 5,252,714 which is incorporated by reference herein for the disclosure of such water soluble polymers), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives, including but not limited to methylcellulose and carboxymethyl cellulose, serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof.
By way of example only, coupling of such water soluble polymers to natural amino acid polypeptides or non-natural polypeptides results in changes including, but not limited to, increased water solubility, increased or modulated serum half-life; increased or modulated therapeutic half-life relative to the unmodified form, increased bioavailability, modulated biological activity, extended circulation time, modulated immunogenicity, modutated physical association characteristics including, but not limited to, aggregation and multitner formation, altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization. In addition, such water soluble polymers optionally have their own biological activity.
1001671 Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, are employed.
1001681 Compounds, (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) presented herein include isotopically-labeled compounds, which are identical to those recited in the various formulas and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygcn, fluorine and chlorine, such as ZH, 'H, 13C, 14C, 15N, 18O, 170, 35S iaF 36CI
respectively. Certain isotopically-labeled compounds described herein, for example those into which radioactive isotopes such as 3H and 14C are incorpbrated, are useful in drug and/or substrate tissue distribution assays.
Further, substitution with isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.
1001691 Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) have asymmetric carbon atoms and can therefore exist as enantiomers or diastereomers. Diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by documented methodologies, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomers, enantioniers, and mixtures thereof are considered as part of the compositions described herein.
1001701 In additional or further embodiments, the compounds described herein (including, but not lirruted to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) are used in the form of pro-drugs. In additional or further embodiments, the compounds described herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned conipounds) are metabolized upon administration to an organism in need to produce a metabolite that is then used to produce a desired effect, including a desired therapeutic effect.
In further or additional embodiments are active metabolites of non-natural amino acids and "modified or unmodified" non-natural amino acid polypeptides.
1001711 The methods and formulations described herein include the use of N-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of non-natural amino acids, non-natural anutto acid polypeptides and modified non-natural amino acid polypeptides. In addition, the non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. 7'he solvated forms of the non-natural amino acids, non-natural amino acid polypeptides and modified non-natural aniino acid polypeptides presented herein are also considered to be disclosed herein.
1001721 Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds).may exist in several tautomeric forms. All such tautomeric. forms are considered as part of the contpositions described.herein. Also, for example all enol-keto forms of any compounds (including, but not linuted to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds) herein are considered as part of the compositions described herein.
1001731 Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing either of the aforementioned compounds) are acidic and form a salt with a pharmaceutically acceptable cation. Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural aniino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds) are basic and accordingly, form a salt with a pharmaceutically acceptable anion. All such salts, including di-salts are within the scope of the compositions described herein and are prepared by documented methodologies. For example, salts are optionally prepared by contacting the acidic and basic entities, in either an aqueous, non-aqueous or partially aqueous medium. The salts are recovered by using at least one of the following techniques_ "filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.
1001741 Pharmaceutically :acceptable salts of the non-natural anlino acid polypeptides disclosed herein are optionally formed when an acidic proton present in the parent non-natural amino acid polypeptides either is replaced by a metal ion, by way of example an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. In addition, the, salt forms of the disclosed non-natural amino acid polypeptides are optionally prepared using salts of the starting materials or intermediates. The non-natural amino acid polypeptides described herein are optionally prepared as a pharmaceutically acceptable acid addition salt (which is a type of a pharmaceutically acceptable salt) by reacting the free base form of non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid. Altematively, the non-natural amino acid polypeptides described herein are prepared as pharmaceutically acceptable base addition salts (which are a type of a pharmaceutically acceptable salt) by reacting the free acid form of non-natural amino acid polypeptides described herein witli a pharniaceutically acceptable inorganic or organic base.
1001751 The type of pharmaceutical acceptable salts, include, but are not limited to: (]) acid addition salts, fornied with inorganic acids=such as hydrochloric acid, hydrobroni.ic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid; succinic acid, malic acid, maleic, acid, fumaric. acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid,. mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-l-carboxylic acid, glucoheptonic acid, 4;4'-methylenebis-(3-hydroxy-2-ene-1 -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base.
Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, troniethamine, N-niethylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydcoxide, sodium carbonate, sodium hydroxide, and the like.
1001761 'rhe corresponding counterions of the non-natural amino acid polypeptide pharmaceutical acceptable salts are analyzed and identified using various methods including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry, or any combination thereof. In addition, the therapeutic activity of such non-natural amino acid polypeptide pharmaceutical acceptable salts are tested using the techniques and methods described in examples 9-17.

1001771 It should be understood that a reference; to a salt includes the solvent addition forms or crystal forms thereof, particularly solvates or. polymorphs..Solvates contain either stoichiometric or non-stoichiometric ainounts of a solvent, and are often formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Polymorphs include the different crystal packing arrangements of the same elem_ental composition of.a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Various factors.such as the recrystallization solvent, rate of crystallization, and.storage temperature are expected to cause a single crystal form,to dominate.
[00178) The screening and characterization of non-natural:amino acid ,polypeptide pltarmaceutical acceptable salts polymorphs and/or solvates is accomplished using a variety of techniquesincluding, but not limited to;
thermal analysis, x-ray diffraction, spectroseopy, vapor soiption; and microscopy. Thermal analysis methods address thermo chemical degradation or thermo physical processes including, but not limited to, polymorphic transitions, and such methods are used to analyze the relationships between polymorphie forms; determine weight loss, to find the glass transition temperature, or for excipient compatibility studies. Such methods include, but are not Iimited.`to, Differential scanning calorimetry (DSC), Modulated Differential Scanning Calorimetry (MDCS), Thermogravimetric analysis (TGA), and Thermogravi-metric and Infrared analysis (TG/IR). X-ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron sources. The various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid state). The various microscopy techniques include, but are not limited to, polarized light microscopy, Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX), Environmental Scanning Electron Microscopy with EDX (in gas or water vapor atmosphere), IR
nucroscopy, and Raman microscopy.

BRIEF DESCRIPTION. OF THE FIGURES

1001791 A better understanding of the features and advantages of the present methods;and compositions. maybe obtained by reference to the following detailed description that sets forth illustrative embodiinents, in which the principles of our methods, coiripositions, devices and apparatuses. are utilized, and the accompanying drawings of :which:
1001801 FIG. I presents a non-linuting schematic representation of the relationship.of certain aspects of the methods, compositions, strategies and techniques described herein.
1001811 FIG. 2 presents a non limiting schematic representation of the mechanism of Fischer indole synthesis.
[001821 FIG. 3 presents illustrative, non-limiting examples of the synthetic methodology used to niake the non-natural aniino, acids described herein.
1001831 FIG. 4 presents illustrative, non-limiting examples of the synthetic methodology used to make the non-natural amino.acids described herein.
1001841 FIG. 5 presents an illustrative, non-limiting example of the synthetic methodology used to make the non-natura( amino acids described herein.
1001851 FIG. 6 presents illustrative, non-limiting examples of the synthetic methodology used to make the non-natural amino acids described herein.

1001861 FIG. 7 presents illustrative, non-limiring,examples of the synthetic methodology used to make the non-natural anuno acids described. herein.
1001871 FIG. 8 presents illustrative, non-limiting.examples of the synthetic niethodology used.to make the non-natural aniino acids described herein.
1001881 FIG. 9 presents illustrative, non-limiting examples of the synthetic methodology used to make the non-natural amino acids described herein.
1001891 FIG. 10 presents illustrative, non-limiting examples of the synthetic methodology used to make the non-natural amino acids described herein.
1001901 FIG. I1 presents illustrative, non-limiting examples of the synthetic methodology used to make the non-natural amino acids described herein.
[00191] FIG. 12 presents illustrative, non-limiting examples of the effect of metal ion on the Fisher indole synthesis.
(00192] FIG. 13 presents illustrative, non-limiting examples of the accelerating effect of nickel metal ion on the synthetic methodology used to make the non-natural anuno acids described herein.
1001931 FIG. 14 presents illustrative, non-limiting examples of the effect of the solvent on the synthetic methodology used to make the non-naniral aminoacids described herein.
1001941 FIG. 15 presents illustrative, non-limiting examples of hydrazine-containing non-natural amino acid reagents used in the synthesis of indole-containing;non-amino acids described.herein.
1001951 FIG. 16; presents illustrative, non-limiting examples of carbonyl-containing and masked carbonyl-containing non-natural amino acid reagents used in the synthesis of indole-eontaining non-natural amino acids described herein.
1001961 FIG. 17 presents illustrative, non-limiting examples of carbonyl-containing and masked carbonyl-containing non-natural amino acid reagents used in the synthesis of indole-containing non-natural amino acids described herein.
1001971 FIG. 18 presents illustrative, non-limiting examples of carbonyl-containing non-natural amino acid reagents used in the synthesis of indole-containing non-natural aniino acids described herein.
1001981 FIG. 19 presents illustrative, non-limiting examples of the post-translational modification of carbonyl-containing non-natural amino acid polypeptides with hydrazine-containing reagents to form modifed indole-containing non-natural amino acid polypeptides.
1001991 FIG. 20 presents illustrative, non-limiting examples of the post-translational modification of hydrazine-containing non-natural amino acid polypeptides with carbonyl-containingreagents to form modifed indole-containing non-natural amino acid polypeptides.
1002001 FIG. 21 presents illustrative, non-limiting examples of carbonyl-containing and masked carbonyl-containing proteins labeling or modification.with hydrazine-containing reagents to form indole-containing non-natural amino:acid proteins.
1002011 FIG. 22 presents illustrative, non-lin-iting exantples of hydrazine-containing proteins labeling or niodification with carbonyl-containing reagents to form indole-containing non-natural amino acid proteins.
1002021 Fig. 23 presents illustrative, non-limiting examples of (A) the modification of non-natural amino acid polypeptides by chemical conversion into carbonyl-containing non-natural amino acid polypeptides and (B) the niodification of non-natural amino acid polypeptides by chemical conversion into hydrazine-containing non-natural amino acid polypeptides. Such non-natural amino acid polypeptides are used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing, and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein.
1002031 FIG. 24A represents illustrative, non-limiting examples of the modification of hydrazine and carbonyl non-natural amino acid.containing polypeptides or proteins to introduce the desired functionality. Such non-natural amino acid polypeptides are used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing, and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein.
1002041 FIG. 24B represents illustrative, non-limiting examples of the reaction of functional group containing polypeptides or proteins with PEG derivatives. Such non-natural amino acid polypeptides are used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing, and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein.
1002051 FIG. 25 presents an illustrative, non-liniiting representation of the use of a bifunctional linker group to link protein or polypeptide containing non-natural amino acid with PEG derivatives through the formation of indole.
1002061 FIG. 26 presents an illustrative, non-linuting examples of the the synthesis a bifunctional linker group containing hydrazine at both ends.
1002071 FIG. 27 presents an illustrative, non-limiting example of the use of a bifunctional linker to form a homodimer of two non-natural amino acids polypeptides.
1002081 FIG. 28 represents illustrative, non-limiting examples of the reaction between branched PEG
containing reagents and carbonyl non-natural amino acid containing polypeptides to form indole modified polypeptides. Such non-natural amino acid polypeptides are used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing, and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein.
1002091 FIG. 29 represents illustrative, non-limiting examples of the reaction between branched PEG
containing reagents and hydrazine non-natural amino acid containing polypeptides to form indole modified polypeptides. Such non-natural anuno acid polypeptides are used in or incorporated into any of the methods, compositions, tecluiiques and strategies for making, purifying, characterizing, and using non-natural amino acids, non-natural amino acid polypeptides and niodified non-natural amino acid polypeptides described herein.
(00210) FIG. 30 represents illustrative, non-limiting examples of PEG
derivatives containing hydrazine and carbonyl groups.

DETAILED DESCRIPTION OF THE INVEN7'ION
I. Introduction 1002111 Recently, an entirely new technology in the protein sciences has been reported, which overcome many of the limitations associated with site-specific modifications of proteins.
Speci6cally, new components have been added to the protein biosynthetic niachinery of the prokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001), Science 292:498-500) and the eukaryote Sacclrornyces cerevisiae (S. cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which has enabled the incorporation of non-natural amino acids to proteins in vivo.
A number of new aniino acids with novel chemical, physical or biological properties, including photoaffinity labels and photoisoinerizable amino acids, keto amino acids, and glycosylated amino acids have been incorporated efficiently and with high fidelity into proteins in E. coli and in yeast in response to the amber codon, TAG, using this methodology. See, e.g., J. W. Chin et al., (2002), Journal of the American Chemical Socie 124:9026-9027 (incorporated by reference in its entirety); J. W. Chin, &
P. G. Schultz, (2002), CheniBioChem 3(11):1135-1137 (incorporated by reference in its entirety); J.
W. Chin, et al., (2002), PNAS
United States of America 99(71):11020-1 1024 (incorporated by reference in its entirety); and, L. Wang, & P. G.
Scliultz, (2002), Chem. Comm., 1-11 (incorporated by reference in its entirety). These studies have demonstrated that it is possible to selectively introduce chemical functional groups that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react efficiently and selectively to form stable covalent linkages.
IL Overview 1002121 FIG. 1 is a non-limiting example of the compositions, methods, techniques and strategies that are described herein. At one level, described herein are the tools (methods, compositions, techniques) for creating and using a polypeptide comprising at least one non-natural amino acid or modified non-natural amino acid with a carbonyl, hydrazine, or heterocycle, including a nitrogen-containing heterocycle group. The carbonyl group includes, but is not limted to, ketones or aldehydes,. Such non-natural amino acids optionally contain further functionality, including but not limited to a desired functionality. Note that the various aforenientioned functionalities are not meant to imply that the members of one functionality can not be classified as menibers of another functionality. Indeed, there will be overlap depending upon the particular circumstances. By way of example only, a water-soluble polymer overlaps in scope with a derivative of polyethylene glycol, however the overlap is not complete and thus both functionalities are cited above.
1002131 As shown in FIG. 1, in one aspect are niethods for selecting and designing a polypeptide to be modified using the methods, compositions and techniques described herein. The new polypeptide is optionally designed de novo, including by way of exantple only, as part of liigh-throughput screening process (in which case numerous polypeptides are designed, synthesized, cliaracterized and/or tested) or based on the interests of the researcher. Alternativly, the new polypeptide is optionally designed based on the structure of a known or partially characterized polypeptide. By way of example only, the Growth Hormone Gene Superfamily (see infra) has been the subject of intense study by the scientific conununity; in one embodiment, a new polypeptide is designed based on the structure of a member or members of this gene superfamily. The principles for selecting which amino acid(s) to substitute and/or modify are described separately herein. The choice of which modification to employ is also described herein, and is used to nieet the need of the experimenter or end user.
Such needs include, but are not limited to, manipulating the therapeutic effectiveness of the polypeptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics, pharmacologics and/or pharmacodynamics of the polypeptide, such as, by way of example only, increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic. half-life, niodulating immunogenicity, modulating biological activity, or extending the circulation time. In addition, such modifications include, by way of example only, providing additional. functionality to the polypeptide, incorporating a tag, label or detectable signal into the polypeptide, easing the isolation properties of the polypeptide, and any combination of the aforementioned modifications.

[002141 Also described herein are non-natural amino acids that have been or are optionally modified to contain a hydrazine, carbonyl, or heterocycle, including a nitrogen-containirig heterocycle group. The carbonyl group iitcludes, but is not limted to, ketones, and aldehydes. Included with this aspect are methods for producing, purifying, characterizing and using such non-natural amino acids. In another aspect described herein are methods, strategies and techniques for incorporating at least one such non-natural amino acid into a polypeptide.
Also included with this aspect are methods for producing, purifying, characterizing and using such polypeptides containing at least one such non-natural amino acid. Also included with this aspect are compositions of and methods for producing, purifying, characterizing and using oligonucleotides (including DNA and RNA) that can be used to produce, at least in part, a polypeptide containing at least one non-natural amino acid. Also included with this aspect are compositions of and methods for producing, purifying, characterizing and using cells that can express such oligonucleotides that can be used to produce, at least in part, a polypeptide containing at least one non-natural amino acid.
1002151 Thus, polypeptides comprising at least one non-natural amino acid or modified non-natural amino acid with a hydrazine, carbonyl, or heterocycle, including a nitrogen-containing heterocycle group are provided and described herein. Carbonyl modified non-natural amino acids include, but are not limted to, ketones and aldehydes. ln certain embodiments, polypeptides with at least one non-natural amino acid or modified tton-natural amino acid with a hydrazine, carbonyl, or heterocycle, including, a nitrogen-containing heterocycle group include at least one co-translational or post-translational modification at some position on the polypeptide. In such emdodiments, the carbonyl modified non-natural amino acids further include, but are not linited to, ketones, and aldehydes. In some embodiments the co-translational or post-translational modification occurs via the cellular machinery (e.g., glycosylation, acetylation,.
acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like), in many instances, such cellular-machinery-based co-translational or post-translational modifications occur at the naturally occurring amino acid sites on the polypeptide, however, in certain embodiments, the cellular-niachinery-based co-translational or post-translational modifications occur on the non-natural amino acid site(s) on the polypeptide.
(00216[ In other embodiments. the post-translational modification does not utilize the cellular machinery, but the futictionality is instead provided by attachment of a molecule (including but not limited to, a desired functionality) comprising a second reactive group to the at least one non-natural amino acid comprising a first reactive group (including but not limited to, non-natural amino acid containing a ketone, an aldehyde, a hydrazine, or a lieterocycle, including a nitrogen-containing heterocycle, functional group) utilizing chemistry metliodology described herein, or others suitable for the particular reactive groups. In certain embodiments, the co-translational or post-translational modification is made in vivo in a eukaryotic cell or in a non-eukaryotic cell.
In certain embodiments, the co-translational or post-translational modification is made in vitro not utilizing the cellular machinery. Also included with this aspect are methods for producing, purifying, characterizing and using such polypeptides containing at least one such post-translationally modified non-natural amino acids.
1002171 Also included within the scope of the methods, compositions, strategies and techniques described herein are reagents capable of reacting with a non-natural amino acid (containing either a carbonyl group, a ketone, a ketoaldehyde, a hydrazine, or: protected forms thereof) that is part of a polypeptid'e so as to produce any of the aforementioned post-translational modifications. In general, the resulting post-translationally modified non-iiatural amino acid will produce at least one indole derivative.
The resulting modified indole-based non-natural amino acid, in some embodiments, undergoes subsequent modification reactions. Also included with this aspect are methods for producing, purifying, characterizing and using such reagents that are capable of any such post-translational modifications of such non-natural aniino acid(s).
1002181 In certain embodiments, the polypeptide includes at least one co-translational or post-translational modification that is made in vivo by one host cell, where the post-translational modification is not normally made by another host cell type. In certain embodiments, the polypeptide includes at least one co-translational or post-translational modification that is made in vivo by a eukaryotic cell, where the co-translational or post-translational modification is not normally made by a non-eukaryotic cell.
Examples of such co-translational or post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition,,phosphorylation, glycolipid-linkage modification, and the like.
In one embodiment, the co-translational or post-translational modification comprises attachment of an oligosaccharide to an asparagine by a GIcNAc-asparagine linkage (including but not limited to, where the oligosaccharide comprises (GIcNAc-Man)2-Man-GIcNAc-G1cNAc, and the like). In another embodiment, the co-translational or post-translational modification comprises attachment of an oligosaccharide (including but not limited to, Gal-GaINAc, Gal-GIcNAc, etc.) to a serine or threonine by a GaINAc-serine, a GaINAc-threonine, a G1cNAc-serine, or a G1cNAc-threonine linkage. Examples of secretion signal sequences include, but are not limited to, a prokaryotic secretion signal sequence, a eukaryotic secretion signal sequence, a eukaryotic secretion signal sequence 5'-optimized forbacterial expression, a novel secretion signal sequence, pectate lyase secretion signal sequence, Omp A secretion signal sequence, and a phage secretion signal sequence. Examples of secretion signal sequences, include, but are not limited to, S"TII
(prokaryotic), Fd GIII and M 13 (phage), Bg12 (yeast), and the signal sequence bla derived from a transposon. In certain embodiments, a protein or polypeptide can comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST
fusion, and/or the like. Also included with this aspect are methods for producing, purifying, characterizing and using such polypeptides containing atleast one such co-translational or post-translational modification. In other embodiments, the glycosylated non-natural amino acid polypeptide is produced in a non-glycosylated form.
Such a non-glycosylated form of a glycosylated non-natural amino acid are optionally produced by methods that include chemical or enzymatic removal of oligosaccharidegroups from an isolated or substantially purified or unpurified glycosylated non-natural amino acid polypeptide; production of ihe non-natural amino acid in a host that does not glycosylate such a non-natural amino acid polypeptide (such a host including, prokaryotes or eukaryotes engineered or mutated to not glycosylate such a polypeptide), the introduction of a glycosylation inhibitor into the cell culture medium in which such a non-natural amino acid polypeptide is being produced by a eukaryote that normally would glycosylate such a polypeptide, or a combination of any such methods. Also described herein are such non-glycosylated forms of normally-glycosylated non-natural amino acid polypeptides (by normally-glycosylated is meant a polypeptide that would be glycosylated when produced ttnder conditions in which naturally-occurring polypeptides are glycosylated). Of course, such non-glycosylated forms of norinally-glycosylated non-natural amino acid polypeptides are optionally in an unpurified form, a substantially purified form, or in an isolated form.
1002191 The non-natural anvno acid polypeptide contains, in alternative embodiments, at least: one, at least two, at least three, at least four, at ]east.five, at least six, at least seven, at least eight, at least nine, or ten or niore non-natural amino acids containing a carbonyl group, a ketone, an aldehyde, a hydrazine, heterocycle, including a nitrogen-containing heterocycle group, or protected forms thereof. The non-natural amino acids can be the same or different, for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different non-natural amino acids. ln certain embodiments, at least one, but fewer than all, of a particular amino acid present in a naturally occurring version of the protein is substituted with a non-natural amino acid.
[00220[ The methods and compositions provided and described herein include polypeptides comprising at least one non-natural amino acid containing a carbonyl group, a ketone, an aldehyde;
a hydrazine, heterocycle, including a nitrogen-containing heterocycle group, or protected or masked forms thereof. Introduction of at least one non-natural amino acid into a polypeptide can allow for the application of conjugation chemistries that involve specific chemical reactions, including, but not limited to, with one or niore non-natural amino acids while not reacting with the commonly occurring 20 aniino acids. Once incorporated, the non-naturally occurring amino acid side chains can also be modified by utilizing chemistry methodologies described herein or suitable for the particular functional groups or substituents present in the naturally encoded amino acid.
1002211 The non-natural amino acid methods and compositions described herein provide conjugates of substances having a wide variety of functional groups, substituents or moieties, with other substances including but not limited to a desired functionality.
1002221 In certain embodiments the non-natural amino acids, non-natural amino acid polypeptides, linkers and reagents described herein, including compounds of Formulas I-XV, and compounds 1-0 are stable in aqueous solution under mildly acidic conditions (including but not lintited to pH of about I to about 6). In other embodiments, such compounds are stable for at least one month under mildly acidic conditions. In other embodiments, such compounds are stable for at least 2 weeks under mildly acidic conditions. In other embodiments, such compounds are stable for at least 5 days under mildly acidic conditions 1002231 In another aspect of the compositions, methods, techniques and strategies described herein are methods for studying or using any of the aforementioned modified or ttttmodified non-natural amino acid polypeptides.
Included within this aspect, by way of example only, are therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy-production, consumer products and/or military uses which would benefit from a polypeptide comprising a modified or unmodified non-natural amino acid polypeptide or protein.
M. Location of non-natural amino acids iu po/ypeptides [002241 The methods and compositions described herein include incorporation of one or more non-natural aniino acids into a polypeptide. One or rnore non-natural amino acids are, in certain embodiments, incorporated at one or more particular positions which does not disrupt activity of the polypeptide. This is optionally achieved by making "conservative" substitutions, including but not limited to, substituting hydrophobic amino acids with non-natural or natural hydrophobic amino acids, bulky amino acids with non-natural or natural bulky amino acids, hydrophilic amino acids with non-natural or natural hydrophilic amino acids) and/or inserting the non-natural amino acid in a location that is not required for activity.
Although such substitions are not known for the non-natural amino acids described herein, the practice of making conservative substitutions within the group of naturally-occurring aniino acids has been documented. Similar approaches are optionally used for the non-natural amino acids described herein.
1002251 A variety of biochemical and structural approaches can be employed to select the desired sites for substitution with a non-natural amino acid within the polypeptide. Any position of the polypeptide chain is suitable for selection to incorporate a non-natural amino acid, and selection is optionally based on rational design or by random selection for any or no particular desired purpose.
Selection of desired sites is optionally based on producing a non-natural amuio acid polypeptide (which is optionally further nwdified or remain unmodified) having any desired property or activity, including but not liinited to agonists, super-agonists, partial agonists, inverse agonists, antagonists, receptor binding modulators, receptor, activity modulators, modulators of binding to binder partners, binding partner activity modulators, biitding partner coiiformation modulators, dimer or multimer formation, no change to activity or property compared to the native molecule, or manipulating any physical or chemical property of the polypeptide such as solubility, aggregation, or stability. For example, locations in the polypeptide required for biological activity of a polypeptide can be identified using methods includ'uig, but not liniited to, point mutation analysis, alanine scanning or homolog scanning methods. Residues other than those identified as critical to biological activity by methods including, but not limited to, alanine or homolog scanning mutagenesis are good candidates for substitution with a non-natural amino acid depending on the desired activity sought for the polypeptide. Altematively, the sites identified as critical to biological activity are also good candidates for substitution with a non-natural amino acid, again depending on the desired activity sought for the polypeptide. Another altemative would be to simpty make serial substitutions in each position on the polypeptide chain with a non-natural amino acid and observe the effect on the activities of the polypeptide.
Any means, technique, or method for selecting a position for substitution with a non-natural amino acid into any polypeptide is suitable for use in the methods, techniques and compositions described.herein.
1002261 The structure and activity of naturally-occurring mutants of a polypeptide that contain deletions can also be examined to deterniine regions of the protein that are likely to be tolerant of substitution with a non-natural amino acid. Once residues that are likely to be intolerant to substitution with non-natural amino acids have been eliminated, the impact of proposed substitutions at each of-the remaining positions can be examined using methods including, but not limited to, the three=dimensional structure of the relevant polypeptide, and any associated ligands or binding proteins. X-ray crystallographic and NMR
siructures of many polypeptides are available in the Protein Data Bank (PDB, www.rcsb.org), a centralized database containing three-dimensional structural data of large molecules of proteins and nucleic acids, one can be used to identify amino acid positions that can be substituted with non-natural amino acids. . In addition, models are optionally made investigating the secondary and tertiary structure of polypeptides, if three-dimensional structural data is not available. Thus, the identity of amino acid positions that are available for substitution with non-natural amino acids is readily obtained.
1002271 Exemplary sites of incorporation of a non-natural amino acid include, but are not limited to, those that are excluded from potential receptor binding regions, or regions for binding to binding proteins or ligands, are fully or partially solvent exposed, have minimal or no hydrogen-bonding inteiactions with nearby residues, are minimally exposed to nearby reactive residues, and/or are in regions that are highly flexible as predicted by the three-diniensional crystal structure of a particular polypeptide with its associated receptor, ligand or binding proteins.
1002281 A wide variety of non-natural amino acids are optionally substituted for, or incorporated into, a given position in a polypeptide. By way of example, a particular non-natural amino acid is selected for incorporation based on an examination of the three dimensional crystal structure of a polypeptide, with its associated ligand, receptor and/or binding proteins, a preference for conservative substitutions 1002291 In one embodiment, the methods described herein include incorporating into the polypeptide the non-natural amino acid, where the non-natural amino acid comprises a first reactive group; and contacting the polypeptide with a molecule (including but not limited to a desired functionality) that coniprises a second reactive group. In certain embodiments, the first reactive group is a carbonyl moiety and the second reactive group is a hydrazine moiety, whereby an indole linkage is formed. In certain embodiments, the first reactive group is a hydrazine moiety and the second reactive group is carbonyl moiety, whereby an indole linkage is formed.
1002301 In some cases, the non-natural amino acid substitution(s) or incorporation(s) will be combined with other additions, substitutions, or deletions within the polypeptide to affect other chemical, physical, pharmacologic and/or biological traits. In some cases, the other additions, substitutions or deletions increase the stability (including but not limited to, resistance to proteolytic degradation) of the polypeptide or increase affinity of the polypeptide for its appropriate receptor, ligand and/or binding proteins. In some cases, the other additions, substitutions or deletions increase the solubility (including but not limited to, when expressed in E.
coli or other host cells) of the polypeptide. In some embodiments sites are selected for substitution with a naturally encoded or non-natural anvno acid in addition to another site for incorporation of a non-natural amino acid for the purpose of increasing the polypeptide solubility following expression in E. coli, or other recotnbinant host cells. In some embodiments, the polypeptides comprise another addition, substitution, or deletion that modulates affinity for the associated ligand, binding proteins, and/or receptor, modulates (including but not limited to, increases or decreases) receptor dimerization, stabilizes receptor dimers, niodulates circulating half-life, modulates release or bio-availability, facilitates purification, or improves or alters a particular route of administration. Siniilarly, the non-natural amino acid polypeptide can comprise chemical or enzyme cleavage sequences, protease cleavage sequences, reactive groups, antibody-binding doniains (including butnot linuted to, FLAG or poly-His) or other affinity based sequences (,including but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to, biotin) that improve detection (including but r(ot limited to, GFP), traasport thru tissues or cell membranes, prodrug release or activation, size reduction,purification or other traits of the polypeptide.
IV. Growth Horn:one Supergene Family as Exemplar 1002311 The methods, compositions, strategies and techniques described herein are not limited to a particular type, class or family of polypeptides or proteins. Indeed, virtually any polypeptide is optionally designed or modified to include at least one modified or unmodified non-natural amino acids described herein. By way of example only, the polypeptide is homologous to a therapeutic protein.
1002321 Thus, the following description of the growth hormone (GH) supergene family is provided for illustrative purposes and by way of example only and not as a limit on the scope of the methods, compositions, strategies and techniques described herein. Further, reference to GH
polypeptides in this application is intended to use the generic term as an example of any member of the GH supergene family. Thus, it is understood that the modifications and chentistries described herein with reference to GH
polypeptides or protein can be equally applied to any member of the GH supergene family, including those specifically listed herein.
1002331 The following proteins include those encoded by genes of the growth hormone (GH) supergene family (Bazan, F., Immunology Today 11: 350-354 (1990); Bazan, J. F. Science 257: 410-411 (1992); Mott, H. R. and Campbell, I. D., Current Opinion in Structural Biology 5: 114-121 (1995);
Silvennoinen, 0. and Ihle, J. N., SIGNALLING BY THE: HGMATOROIGTIC CYTOKINE RECCPTOR5 (1996)): growth hormone, prolactin, placental lactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, I.L-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor, leukeniia inhibitory factor, alpha interferon, beta interferon, epsilon interferon, gamma interferon, omega interferon, tau interferon, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macropliage colony stimulating factor (M-CSF) and cardiotrophin-l (CT-1) ("the GH supergene family"). It is anticipated that additional members of this gene family will be identified in the future through gene cloning and sequencing. Members of the GH supergene family have similar secondary and tertiary structures, despite the fact that they generally have liniited amino acid or DNA sequence identity.
The shared structural featuresallow new members of the gene family to be readily identified and the non-natural amino acid methods and conipositions described herein sinularly applied.
1002341 Structtues of a number of cytokines, including G-CSF (Zink et al., FEBS Lett. 314:435 (1992); Zink et al., Biochemistry 33:8453 (1994); Hill et al., Proc.Natl.Acad.Sci.USA 90:5167 (1993)), GM-CSF (Diederichs, K., et al. Science 154: 1779-1782 (1991); Walter et al., J. Mol. Biol.
224:1075-1085 (1992)), IL-2 (Bazan, J. F.
and McKay, D. B., Science 257: 410-413 (1992); IL-4 (Redfield et al., Bioc{remistty 30: 11029-11035 (1991);
Poweis et al., Science 256:1673-1677 (1992)), and IL-5 (Milbum et al., Nature 363: 172-176 (1993)) have been determined by X-ray diffraction and NMR studies and show striking conservation with the GH stnicture, despite a lack of significant primary sequence homology. IFN is considered to be. a member of this family based upon modeling and other studies (Lee et al., J. Interferon Cytokine Res. 15:341 (1995); Murgolo et al., Proteins 17:62 (1993); Radhakrishnan et al., Structure 4:1453 (1996); Klaus et al., J. Mol.
Biol. 274:661 (1997)). A large number of additional cytokines and growth factors including ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), thrombopoietin (TPO), oncostatin M, macrophage colony stimulating factor (M-CSF), IL-3, IL-6, IL-7, IL-9, IL-l2, IL-13, IL-15, and granulocyte-colony stimulating factor (G-CSF), as well as the IFN's such as alpha, beta, omega, tau, epsilon, and gamma interferon belong to this family (reviewed in Mott and Campbell, Current Opinion in Structural Biology 5: 114-121 (1995);
Silvennoinen and Ihle (1996) SIGNALLING E3Y THE HEMATOPOIETIC CYTOKiNE RECEPTORS). All of the above cytokines and growth factors are now considered to comprise one large gene family.
(002351 In addition to sharing similar secondary and tertiary structures, members of this family share the property that they must oligomerize cell surface receptors to activate intracellular signaling pathways. Some GH
family members, including but not linuted to; GH and EPO, bind a single type of receptor and cause it to form homodimers. Other family members, including but not limited to, IL-2, IL4. and IL-6, bind more than one type of receptor and cause the receptors to form heterodimers or higher order aggregates (Davis et al., (1993) Science 260: 1805-1808; Paonessa et al., 1995) EMBO J. 14: 1942-1951; Mott and Campbell, Current Opinia: in Sti-uctura! Biology 5: 114-121 (1995)). Mutagenesis studies have shoNvn that, like GH, these other cytokines and growth factors contain multiple receptor binding sites, typically two, and bind their cognate receptors sequentially (Mott and Campbell, Current Opinion in Structural Biology 5: 114-121 (1995); Matthews et al., (1996) Proc. Natl. Acad. Sci. USA 93: 9471-9476). Like GH, the primary receptor binding sites for these other family members occur priinarily in the four alpha helices and the A-B loop.
The specific amino acids in the helical bundles that participate in receptor binding differ amongst the family niembers. Most of the cell surface receptors that interact with niembers of the GH supergenc family are struchually related and comprise a second large multi-gene family. See, e.g. U.S. Patent No. 6,608,183, which is herein incorporated by reference for the description of the GH supergene family.

1002361 A general conclusion reached from mutational studies of various members of the GH supergene family is that the loops joining the alpha helices generally tend to not be involved in receptor binding. In particular the short B-C loop appears to be non-essential for receptor binding in most, if not all, family members. For this reason, the B-C loop are optionally substituted with non-natural amino acids as described herein in members of the GH supergene family. The A-B loop, the C-D loop (and D-E loop of interferon/ IL-10-like members of the GH superfamily) are also opttionally substituted with a non-natural amino acid. Amino acids proximal to helix A and distal to the final helix also tend not to be involved in receptor binding and also are optional sites for introducing non-natural amino acids. In sonie embodiments, a non-natural amino acid is substituted at any position within a loop structure including but not limited to the first 1, 2, 3, 4, 5, 6, 7, or more amino acids of the A-B, B-C, C-D or D-E loop. In some embodiments, a non-natural aniino acid is substituted within the last 1, 2, 3. 4, 5, 6, 7, or more amino acids of the A-B, B-C, C-D or D-E loop.
1002371 Certain members of the GH family, including but not limited to, EPO, IL-2, IL-3, IL-4, IL-6, IFN, GM-CSF, TPO, IL-10, II,-12 p35, IL-13, IL-15 and beta interferon contain N-linked and/or 0-linked sugars. The glycosylation sites in the proteins occur almost exclusively in the loop regions and not in the alpha helical bundles. Because the loop regions generally are not involved in receptor binding and because they are sites for the covalent attachment of sugar groups, they are useful sites for introducing non-natural amino acid substitutions into the proteins. Amino acids that comprise the N- and 0-linked glycosylation sites in the proteins are sites for non-natural amino acid substitutions because these amino acids are surface-exposed. Therefore, the natural protein tolerates:bulky sugar groups attached to the proteins at these sites and the glycosylation sites tend to be located away from the receptor binding sites.
1002381 Additional members of the GH gene family are likely to be discovered in the future. New members of the GH supergene family can be identified through computer-aided secondary and tertiary structure analyses of the predicted protein sequences, and by selection techniques designed to identify molecules that bind to a particular target. Members of the GH supergene family typically possess four or five amphipathic helices joined by non-helical amino acids (the loop regions). In some embodiments, the proteins contain a hydrophobic signal sequence at their N-terminus to promote secretion from the cell. Such later discovered niembers of the GH
supergene family also are included within the methods and compositions described herein.
V. Non-nalura! Amino Acids 1002391 The non-natural amino acids used in the methods and compositions described herein have at least one of the following four properties: (1) at least one functional group on the sidechain of the non-natural amino acid has at least one characteristic and/or activity and/or reactivity orthogonal to the chemical reactivity of the 20 common, genetically-encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutanuc acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, 1ryptophan, tyiosine, and valine), or at least orthogonal to. the chemical reactivity of the naturally occurring amino acids present in the polypeptide that includes the non-natural amino acid; (2) the introduced non-natural amino acids.are substantially chemically inert toward the 20 conunon, genetically-encoded amino acids; (3) the non-natural amino acid can be stably incorporated into a polypeptide, preferably with the stability conunensurate with the naturally-occurring amino acids or under typical physiological conditions, and further prcferably such incorporation can occur via an in vivo system; and (4) the non-natural amino acid includes a carbonyl group, a ketone group, an aldehyde group, a hydrazine group, heterocycle, including a nitrogen-containing heterocycle group (including, an indolyl group), or a functional group that can be transformed into an carbonyl group, a ketone group, an aldehyde group, a hydrazine group, heterocycle, including a nitrogen-containing heterocycle group (including an indolyl group), by reacting with a reagent, preferably under conditions that do not destroy the biological properties of the polypeptide that includes the non-natural amino acid (unless of course such a destruction of biological properties is the purpose of the modification/transformation), or preferably where the transformation can occur under aqueous conditions at a pH between about I and about 6, or preferably. where the reactive site on the non-natural amino acid is an electrophilic site. Illustrative, non-limiting examples of amino acids that satisfy these four properties for non-natural amino acids that can be used with the compositions and methods, described herein are presented in FIGS.
15-18. Any number of non-natural amino acids can be introduced into the polypeptide:
1002401 Note that, even though FIGS. 15, 16, and 18 present amino acids having a substituted carbocyclic aryl sidechain, it is to be understood that these FIGS. also disclose substituted heteroaryl sidechains. By way of exainple, each of the compounds of FIG. 15 has a substittited phenyl sidechain; however, in some embodiments, the phenyl group is replaced by a pyridinyl group, a pyrmidinyl group, a pyrazinyl group, a thiofuranyl group, or a fttranyl group. Thus, the carbocyclic aryl sidechains are merely illustrative exaniples of the variety of aromatic groups included within the present disclosure.
1002411 Non-natural amino acids optionally also include a carbonyl group or a protected or masked groups that can be transformed into a carbonyl group, a carbonyl group after deprotection of the protected group or unmasldng of the masked group, a hydrazine group or a protected or masked group that can be transformed into a hydrazine group.
1002421 Non-natural amino acids -that are optionally used in the methods and compositions described herein include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and/or photoisomerizable amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or other polyethers, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active anuno acids, amino thioacid containing amino acids, and amino acids comprising one or niore toxic moiety.
1002431 In some embodiments, non-natural amino acids comprise a saccharide moiety. Examples of stich amino acids include N-acetyl-L-glucosanunyl-L-serine, N-acetyl-L-galaetosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine and 0-mannosantinyl-L-serine. Examples of such amino acids also include examples where the naturally-occurring N- or 0- linkage between the amino acid and the saccharide is replaced by a covalent linkage not commonly found in nature. -including but not limited to, an alkene, an oxime, a thioether, an amide, a heterocycle, including a nitrogen-containing heterocycle, a carbonyl and the like. Examples of such amino acids also include saccharides that are not commonly found in naturally-occurring proteins such as 2-deoxy-glucose, 2-deoxygalactose and the like.

1002441 The chenucal inoieties incorporated into polypeptides via incorporation of non-natural amino acids into such polypeptides offer a variety of advantages and manipulations of the polypeptides. For example, the unique non-natural amino acids (including but not limited to, amino acids with benzophenone and arylazides (including but not limited to, phenylazide side chains), for example, allow for efficient in vivo and in vitro photocrosslinking of protein. Examples of photoreactive non-natural anuno acids include, but are not limited to, p-azido-phenylalanineand p-benzoyl-phenylalanine. The polypeptide with the photoreactive non-natural amino acids is then optionally crosslinked at will by excitation of the photoreactive group-providing temporal control.
In a non-limiting example, the methyl group of a non-natural amino is substituted with an isotopically labeled, including but not limited to, with a methyl group, as a probe of local structure and dynamics, including but not limited to, with the use of nuclear magnetic resonance and vibrational'spectroscopy.

A. Structure and Synthesis of Non-Natrtral Amino Acids: Hydrazine, Hydraziue-like, Masked Hydrazine, asd Protected Hydrazine Groups 1002451 Non-natural amino acids containing a hydrazine group allow for reaction with a variety of carbonyl and carbonyl equivalent groups to form conjugates via indole linkage. Thus, in certain embodiments described herein are non-natural amino acids with sidechains comprising a hydrazine group, a hydrazine like group (which has reactivity similar to a hydrazine group and is structurally similar to a hydrazine group), a niasked hydrazine group (which can be readily converted into a hydrazine group), or a protected hydrazine group (which has reactivity similar to a hydrazine group upon deprotection). Such amino acids include amino acids having the structureofFormula (1):

R} ~ JR
H R;A,B

0 (I), wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene;
lower cycloalkylene, substituted lower cycloalkyl"ene, lower alkenylene, substituted lower alkenylene, alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)Z-, -OS(O)z-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNO2)N(R')-, -N(R')C(NCOOR')N(R')-, -N(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-;. and each R' is independently.H, alkyl, or substituted alkyl;

N Ny N ~N N
\NHt N%~ I ~NH= i ~ . .
~~ I NH= NH=
NHp r I ( (y (\ N N
Jis~', or R is H, lower alkenylene, substituted lower alkenylene,,alkynylene;
substituted alkynylene; carbonyl, or substituted. carbonyl;
R' is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
R2.is OH, an ester protecting group, resin, at least.one amino acid, polypeptide,, or polynucleotide;
each of R3 and R 4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 taken together or two R3 groups taken together optionally form a cycloalkyl or a heterocycloalkyl;
or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising a hydrazine group, a protected hydrazine group or a masked hydrazine group;

It should be noted that, where I appears in structure J, this means that J can be attached to B and R at any position. As a non-limiting example, where J is a hydrazinephenyl derivative, B and J are optionally positioned around the ring, as illustrated below:

N..NH2 - \ ~ N`NHz N.NHZ R q N_NH2 R NNH2 R NNH2 R N,NHZ
R g~ ~g I

H N
H R
/ I N' NH2 /~ N.NH2 g/' NNH2 g/ N,NH2 g/ N.NH2 ,g &,N~NH, g \ R g~ ~ I R R~ ~ R R

It should also be further noted that the ring is optionally further optionally substituted. In addition it should be noted that at least one of the adjacent substituent to hydrazine group is hydrogen. Such non-natural amino acids are optionally in the form of a salt, or incorporated into a non-natural amino acid polypeptide, polynier, polysaccharide,. or a polynucleotide and optionally post translationally modified.
1002461 In onc embodiment, both A and B are bonds, each R3 is H and R, is H.
In a further embodiment, each of R, and R2 are at least one amino acid. In a further embodiment, each of R, and R_, are at least two amino acids. In a further embodiment, each of Ri and R,-are at least three amino acids. In a further embodiment, each of Ri and R2 are at least four amino acids. In a further embodiment, each of R, and R2 are at least five amino acids. In a further embodiment, each of R, and R, are at least eix amino acids.
1002471 In certain embodiments, compounds of Formula (I) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Fomiula (I) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (I) are stable for at least 5 days under nuldly acidic conditions. In certain embodiments, such acidic conditions are pH about 2 to about 8.

1002481 In addition, the following amino acids having the structure of Formula (11) are included:
Ra H AB , \ Ra R' N~ NN.Ra R N
0 R, . (II) wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, substitutedalkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenyiene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or sutistituted alkylene)-, -C(O)-,. -NS(O)2-, -OS(Q)2-, -C(O)-(alkylene or substituted alkylene)-; -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, =N-O-(alkylene or substituted alkylene), -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-;,and each R' is independently H, alkyl, or substituted alkyl;
R, is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
R3 and R4 is independently selected from the group consisting of hydrogen or from an amine protecting group, including, but not limited to, H
~YO1_~CCI3 .,IryO,,,~/' O ~ O O O OzN CZHS
/~
CN
r-rl yO~CH ' ~yC%CN =~~ = -C~ W3 or ~~0./~SiMe3 ~,O ~ 1 O ~ O H3C ,. ~ O ~ CH; O ; and each Re is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, CN, NOZ, -N(R.')Z, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2 or 3 and each R' is independently H, alkyl, or substituted alkyl.
Such non-natural amino acids are optionally 'in the form of a salt, or are incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

1002491 In one.embodiment; both A and B are bonds. In a further embodiment, each of R1 and R,_ are at least one amino acid. In a further embodiment, each of Ri and R2 are at least two amino acids. In a further embodinient, each of R, and R2 are at least three amino acids. In a further embodiment, each of Ri and R2 are at least four amino acids. In a further embodiment, each of R, and R2 are at least five amino acids. In a further embodiment, each of R, and R, are at least eix amino acids.
1002501 In addition, the following, amino acids having the structure of Formula (Ill) are included:

R' N (CRaa)n.--B N-N,R

0 R2 (III), wherein:
B is optional, and wlien present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -N(R')-, -S-, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)Z-, -OS(O)Z-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-. -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSI`T(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)0-, -S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')rN=I`'-, and -C(R')2-N(R')-N(R')-; -S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')Z-N=N-, and -C(R')2-N(R')-N(R')-; and each R' is independently H, alkyl, or substituted alkyl;
R' is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
R3 and R4 is independently selected from the group consisting of hydrogen or from an amine protecting group, including, but not limited to, i ~ H
; \
~\ yO.V CCl3 ~~0~ ~~O \ ~ fY0 ~ l C
j( O ~ O O O OZN CZHS
~\
O-~.~,( CH3 ~
-~~ (I CN ~~/ iCH3 Ur ~
. ~ O./~ SiMe3 O ~
~ p ~ 1II 1~ O H3C CHs CN O ,~=c CH3 O ; and each R, is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')2, -C(G)R', -C(O)N(R')2, -OR', and -S(O)kR',:where k is 1, 2 or 3 and each R' is independently H, alkyl, or substituted alkyl; and nis0to8.
Such non-natural amino acids niay be in the form of a salt, or niay be incorporated into a non-natural amino acid polypeptide, polynier, polysaccharide, or a polynucleotide and optionally post translationally modified.

1002511 In yet another embodiment is a conipound of Formula (II) having the structure:
R.
(CH2)nXI(CO)yX2(CI-12)m'$ R4 Rl-, N R2 13 R~ I/ N.R3 H R4O R. H

wherein, n is 6, 1, 2, 3, or 4; m is 0, 1, 2, 3, or 4; provided that n + m is 1, 2, 3, 4, or 5; Xi and X2 are independently a bond, 0, or NH; and y is.0 or 1.
1002521 In addition, the following.amino acids are included:
H
HN N, NH2 HN O N N
~`H .NHZ H N`NH
H N OH HZN OH H2N H s z 0 0 0 , and , .
H
II NNHZ
N a HNJ O
HN--'Z~'N' CN

0 , wherein such compounds are optionally amino protected and carboxyl protected, or a salt thereof, or niay be incorporated into a non-natural amino acid polypeptide, polyiner, polysaccharide, or a polynucleotide and optionally post translationally modified.
1002531 In.addition, the following amino acids having the.structure of Formula (IV) aie included:
Ra H
Ra N, N" R4 I

Rl,,N R 2 H
0 (IV) wherein:
R' is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
R3 and R4 are independently selected from the group consisting of hydrogen or from an amine protecting group, including, but not limited to, i /Y OvCCij J~O I ~=C H
0 0 ; 0 O OZN CzHs O CN
~ S ~/
~ O y ~CH3 S=C~ II ~_ ;GH3 or ~0,tiSiMey ~ 0 H3C CH3 ; SSS CN 0 CH3 0 0 ; and each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, CN, NO2, -N(R'),,, -C(O)R', -C(O)N(R'),, -OR', and -S(O)kR', where k:is 1,2 or 3 and each R' is independently H, alkyl, or substituted alkyl.
Such non-naturai anuno acids are optionally in the form of a salt, or incorporated into a non-natural 'amino acid polypeptide, polynier, polysaccharide, or a polynucleotide and optionally post translationally modified.
1002541 In addition, the following amino acids are included:

N H ~ H H
NH2 I N, N/ \ N_N~ N- N
H
\ \ ~ \
OH OH OH OH

O O O , O
CN

I NNCN HZN N, I
OH OH
HyN
o ; and o wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or optionally incorporated into a non-natural aniino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post.translationally modified.

B Structure and Synthesis of Non-Natttral Amino Acids: carbonyl, carbonyl-like, Masked carbonyl,, and Protected carbonyl Grottps [002551 Amino acids with an electrophilic reactive group allow for a variety of reactions to link molecules via various chemical reactions, including, but not limited to, nucleophilic addition reactions. Such electrophilic reactive groups include a carbonyl group (including a keto- or aldehyde group), a carbonyl-like group (which has reactivity similar to a carbonyl group and is structurally similar to a carbonyl group), a masked carbonyl group (which can be readily converted into a carbonyl group), or a protected carbonyl group (which has reactivity similar to a carbonyl group upon deprotection). Such amino acids include amino acids having the structure of Formula (V):

R3 A,_B/J\R
R~\ R2 H
0 (v), wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene,, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k(alkylene or substituted alkylene)-, where k is 1, 2, or 3, -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, and -N(R')CO-(alkylene or substituted alkylene)-, where each R' is independently H, alkyl, or substituted alkyl;

SR' R" O+N Rõ
O R" Rõ
OR"
~

J - ~s ~ O
o > , ~~
R" R" R" R"
R O S s s N ; or R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
each R" is independently H, alkyl, substittited alkyl, or a protecting group, or when more than one R" group is present, two R" optionally form a heterocycloalkyl;
Ri is 14, an anvno protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group,.including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group;
or the -J-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group;
with a proviso that when A is phenylene and each R3 is H, B is present; and that when A is -(CHZ)4- and each R, is H, B is not -NHC(O)(CH2CH2)-; and that when A and B are absent and each R3 is H, R is not methyl.
Such non-natural amino acids are optionally in the form of a salt, or incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.
1002561 In certain embodiments, compounds of Formula (V) are stable in aqueous solution for at least I
month under mildly acidic conditions. In certain embodiments, compounds of Formula (V) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiinents, compound of Formula (V) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH of about 2 to about 8.

(00257( In certain embodiments of compounds of Formula (V), B is lower alkylene, substituted lower alkylene, -O-(alkylene or substituted alkylene)-, arylene,. substituted arylene, heteroarylene, substituted heteroarylene, -C(O)-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-, or -S(O)2(alkylene or substituted alkylene)-. ]n certain embodiments of compounds of Fomtula (1), B is -O(CH2)-, -CH=N-, -CH=N-NH-, -NHCH2-, -NHCO-, -C(O)-,_ -C(O)-(CHz)-,, -CONH-(CHZ)-, -SCH2-, -S(=O)CH2-, or -S(O)2CHZ-. In certain embodiments of compounds of Formula (V), R is Ci_6 alkyl or cycloalkyl. In certain embodiments of compounds of Formula (V) R is -CH3, -CH(CH3)2, or cyclopropyl. In certaui embodiments of compounds of Formula (V), R, is H, tert-butyloxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula (V), Ri is a resin, at least one amino acid, polypeptide, or polynucleotide. In certain embodiments of compounds of Forniula (V), R2 is OH, 0-methyl, O-ethyl, or 0-t-butyl. In certain embodiments of compounds of Formula (V), R2 is a resin, at least one amino acid, polypeptide, or polynucleotide. In certain embodiments of compounds of Formula (V), R2 is a polynucleotide. In certain embodiments of conYpounds of Formula (V), R2 is ribonucleic acid (RNA). In certain embodiments of compounds of Formula (V), R2 is tRNA. In certain embodiments of compounds of Formula (V), ibe tRNA specifically recognizes a selector codon.
In certain embodiments of compounds of Formula (V) the selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon.
In certain embodiments of compounds of Formula (V), R2 is a suppressor tR.N'A.

1002581 In certain embodiments of compounds of Formula (V), B is selected from the group consisting of:
(i) A is substituted lower alkylene, C4-arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2-, -N'S(O)z-, -OS(O)z-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R')-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -N(R')C(S)-, -S(O)N(R'.), -S(O)zN(R'), -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O)ZN(R')-, -C(R')=N-N(R')-,~-C(R')Z-N=N-, -N(R')C(NCN)N(R')-, -N(R')C(NNOZ)N(R')-, -N(R')C(NCOOR')N(R')-, and -C(R')2-N(R')-N(R' )-;
(ii) A is optional, and when present is substituted lower alkylene, C4-arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)Z-, -NS(O)Z-, -OS(O)2-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-1 -N(R')-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -N(R')C(S)-, -S(O)N(R'), -S(O)2N(R'), -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O)2N(R')-, -C(R')=N-N(R')-, -C(R')2-N=N-, -N(R')C(NCN)N(R')-, -N(R')C(NNOZ)N(R')-, -N(R')C(NCOOR')N(R')-, and -C(R')2-N(R')-N(R')-;
(iii) A is lower alkylene;
B is optional, and when present is a divalent littker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -O-(alkylene or substituted alkylene)-, -C(O)-(alkylene or substituted alkylene)-, and -CON(R')-(alkylene or substituted alkylene)-, and (iv) A is pbenylene;
B is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -O-(alkylene or substituted alkylene)-, -C(O)-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, and -N(R')CO-(alkylene or substituted alkylene)-;

o c O R" > ~,~ ~
J is o/ ~
~ f Rõ R" Rõ Rõ
R O s s s ;or each R' is independently H, alkyl, or substituted alkyl;
each R" is independently H, alkyl, substituted alkyl, or'a protecting group, or when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
R, isoptional, and when present, is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group,.resin, at least one amino acid, polypeptide, or polynucleotide; and each R3 and Ra is independently H, halogen, lower alkyl, or substituted lower alkyl;
R' is H, alkyl, or substituted alkyl;
R is H. alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl.

1002591 In addition, amino acids having the structure of Formula (VI) are included:
O
B'k R
Rl-, N RZ
H
O (VI), wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optioual, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)z-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -N(R.')C(S)NR'-(alkylene or. substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, -N(R')C(NCN)NR'-(alkylene or substituted alkylene), -N(R')C(NNOZ)NR'-(alkylene or substituted alkylene), -N(R')C(NCOOR')NR'-(alkylene or substituted alkylene), and -C(R')2-N(R')-I`T(R')-, where each R' is independently H, alkyl, or substituted alkyl;
R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
R, is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
with a proviso that when A is phenylene, B is present; and that when A is -(CH2)4-, B is not -NHC(O)(CH2CH2)-; and that when A and B are absent, R is not methyl. Such non-natural amino acids are optionally in the form of a salt, or incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.
1002601 In addition, amino acids having the structure of Formula (VII) are included:
Ra Ra By R
O
Ra Ra R, ~, N R2 H
0 (VII), wherein:
B is a linker selected from the group consisting of lower alkylene, substituted lower alkylene,.lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)z-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -S(O)kN(R')-(alkylene or substituted alkylene)-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -N(R')C(S)NR'-1.0 (alkylene or substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, -N(R')C(NCN)NR'-(alkylene or substituted alkylene), -N(R')C(NNO,)NR'-(alkylene or substituted alkylene), -N(R')C(NCOOR')NR'-(alkylene or substituted alkylene), and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
Ri is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
each R. is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, NO2, CN, -N(R')2, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where each R' is independently H, alkyl, or substituted alkyl; or at least two R, taken together form a heterocycle, heteroaryl or aryl.
Such non-natural amino acids are optionally in the form of a salt, or incorporated into a non-natural aniino acid polypeptide, polymer, polysaccharide,'or a polynucleotide and optionally post translationally modified.
1002611 In addition, the following amino acids are included:

O Nj O
O ~) Q
O O~(\ O eOH N~% -NH 0 ~ O I~ I~ O II ~\/
H2N OH H~ OH O HzN HyN OH
O O O O
O SJ, NJ, O

HzN COOH O O
F ~
i0 F
H2N OH HzN OH
0 and 0 Such non-natural anuno acids are optionally amino protected group, carboxyl protected and/or in the form of a salt, or incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.
1002621 In addition, the following amino acids having the structure of Formula (VIII) are included:
O
(CRa)n\Blj~ R

Ri-, N RZ
H
0 (VIII) wherein - B is optional, and when present is a linker selected from the. group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenytene, lower heteroalkylene, substituted lower heteroalkylene, arylene; substituted arylene, heteroarylene; substituted he,teroarylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -N(R')C(S)NR'-(alkylene or substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, -N(R')C(NCN)NR'-(alkylene or substituted alkylene), -N(R')C(NNO2)NR'-(alkylene or substituted alkylene), -N(R')C(NCOOR')NR'-(alkylene or substituted alkylene), and -C(R')Z-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
R, is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
each R,,is independently selected from the group consisting of H, halogen,.alkyl, substituted alkyl, CN, NO2, -N(R')2, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8;
with a proviso that when A is -(CH2)4-, B is not NHC(O),(CH2CH2)-.-Such non-natural amino:acids are optionally in.the form of a salt, or are incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, ar apolynucleotide and optionally post translationally modified.

1002631 In addition, the following amino acids are included:

~ O O O
p ~ O 000 s HZN OH H2Nf~H Hz H H2N OH H2N OH HZN OH H2N H
O , . ~ O ~ = O
HN p N N"
O
HzN ~.{~N

p O O 0 OH OH
O~

p N" `
~O FO
N (N
NH N
HZN N
HzN H2N F OH

0 OH , 0 OH , p OH , and 0 , wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or are incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post tTanslationally modified.
(002691 In addition, the following amino acids having thestructure,ofFormula (IX) are included:
Z
A" B'-'~ Y

Ri R2-H
0 (IX), wherein, A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene; aralkylene, or substituted aralkylene;
B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylenc, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-,-S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)Z-, -OS(O)Z-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene, or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-(alkylene.or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -N(R')C(S)NR'-(alkylene or substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, -N(R')C(NCN)NR'-(alkylene or substituted alkylene), -N(R')C(NNOZ)NR'-(alkylene or substituted alkylene),-N(R')C(NCOOR')NR'-(alkylene or substituted alkylene), and -C(R')Z-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
R, is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
R2 is OH,. an ester protecting,group, resin,. at least one amino acid, polypeptide, or polynucleotide; and Y and Z are independently selected from the group consisting of -OH, alkyl substituted oxygen, -SH or alkyl substituted sulfur and when Y and Z taken together can form a cycloalkyl ring.
Such non-natural amino acids are optionally in the form of a salt, or are incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.
1002651 In addition, the following amino acids having the structure of Forniula (X) are included:
Ra Ra B'T-z Y
Ra Ra Ri, N Rz H
O (X), wherein, B is optional, and when present is a linker selected fromthe_group consistingof.lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)z-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -N(R')C(S)NR'-(alkylene or substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, -N(R')C(NCN)I`TR'-(alkylene or substituted alkylene), -N(R')C(NNOZ)NR'-(alkylene or substituted alkylene), -N(R')C(NCOOR')NR'-(alkylene or substituted alkylene), and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
R, is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
Each Re is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, CN, NO2i -N(R')2, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3,where each R' is independently H, alkyl, or substituted alkyl; or at least two Ra taken together form a heterocycle, hetoroaryl or aryl; and Y and Z are independently selected from the group consisting of -OH, alkyl substituted oxygen, -SH or alkyl substituted sulfur and when Y and Z taken together can form.a cycloalkyl ring.
Such non-natural amino acids are optionally in the form of a salt, or are incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

(002661 In addition, the following amino acids are included:

\ ~ O \ ___~ I/ sJ ~/ I

H2 H HZN OH HzN H H2N OH

QH
\ I ~ O~ O

O O~
O~.
\ N eOH N/!~O
/ I "
H2N H HZ H ~N H2N OH
O O O
O e ro O~ H~ OH H2N o and wherein such compounds are optionally amino protected, optiotially carboxyl protected, optionally aniino protected and carboxyl protected, or a salt thereof, or are incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.
1002671 In addition, the following amino acids having the structure of Forniula (XI) are included:
Ra Re B.~N,~, ~ R~
Ra Re RI, N R2 H
0 (XI) B is optional, and when present is a linker selected from the group consisting=of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(0)2-, -OS(0)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -N(R')C(S)NR'-(alkylene or substitutcd alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -C(R')=N-, -C(R')=N-N(R')-, -C(R')Z=N=N-, -N(R')C(NCN)NR'-(alkylene or substituted alkylene), -N(R')C(NNO2)NR'-(alkylene or substituted alkylene), -I`'(R')C(NCOOR')NR'-(alkylene or substituted alkylene), and -C(R')Z-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
R is fl, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R, is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
R, is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
Each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, CN, NOZ, -N(R')2, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where each R' is independently H, alkyl, or substituted alkyl; or at least two R, taken together form a heterocycle, hetoroaryl.or aryl; and Y independently selected from the group consisting of OR", NR"R' ; NC(O)R"
where each R" is is independently H, alkyl, substituted alkyl.
Such non-natural amino_acids are optionally in the form of a salt, or are incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, `or a polynucleotide and optionally post translationally modified.
1002681 In addition the following amino acids are included:
O O
N
\ ~ N.N ~ i N.On N=N N,II
H N
I~ ~ o 0 0 o and o wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or are incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.
1002691 In addition, the following amino acids having the structure of Formula (X[I) are included:

R ~
a O
\ ~ x )n Ra ~
R, 'N RZ R4 H O (XIl) R, is H, an amino protecting group, resin, at least one aniino acid, polypeptide, or polynucleotide;
R-is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
Each R. is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl,CN, NOz, -N(R'),, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where each R' is independently H, alkyl, or substituted alkyl; or at least two R0 taken together form a heterocycle, hetoroaryl or aryl;
R3 and R4 are independently H, halogen, CN, NOz, alkyl, substituted alkyl, N(R')2, C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where each R' is independently H, alkyl, or substituted alkyl;
X is C, N, 0, S; with the proviso that when X is 0, or S, R4 cannot be H, halogen, CN, NO2, alkyl, substituted alkyl, N(R')2, C(O)R', -C(O)N(R')2, -OR', and -S(O)kR'; where k is 1, 2, or 3, and n is 0, 1 or 2.

1002701 In addition the following ainino acids are included:
OMe I O / ~ O /.I O Me0 ./ I O
o o I ~ ~

H2N OH H2N OH H2N OH F H~ OH HZN OH H2N OH

O Mo0 O
F
H2N OH HzN OH H2N OH
0 0 and o wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or are incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.
1002711 In addition, the following amino acids having the structure of Formula (XIII) are included:
Z
(CRa)n-0 B /\y Ri, N R2 H
0 (XIII), wherein, B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -O-(alkylene or substituted alkylene)-,, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)Z-, -OS(O)z-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -N(R')C(S)NR'-(alkylene or substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -C(R')=IN'-, -C(R')=N-N(R')-, -C(R')Z-N=N-, -N(R')C(NCN)NR'-(alkylene or substituted alkylene), -N(R')C(NNOZ)NR'-(alkylene or substituted alkylene), -N(R')C(NCOOR')NR'-(alkylene or substituted alkylene), and -C(R')Z-N(R')-N(R')-, where each R' is independently N, alkyl, or substituted alkyl;
R, is 11, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
each R, is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where each R' is independeutly H, alkyl, or substituted alkyl; and n is 0 to 8, and Y and Z are independently selected from the group consisting of -OH, alkyl.
substituted oxygen, -SH or alkyl substituted sulfur and when Y and Z taken together can form a cycloalkyl ring.
Such non-natural amino acids are optionally in the form of a salt, or are incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

(00272) In addition, the following amino acids are included:
o o_~
O O o PO ~
pp Oy NH NH NH NH

H2N H H2N H H2N H H2N~O HZN~OH HZN OH HN OH
O O O O

0. 7 \

O O O
H2N H H2N H H2 OH H2N OH H2NI~OH H2N~OH ~N OH
, o 0 0 (0~ o and o wherein such conipounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or are incorporated into a non-natural ainino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.
1002731 In addition, the following atnino acids having the>structure of Formula (XIV) are included:
R
CRa)k, g ~N /Y

R, .N R2 H 0 (XIV), wherein, B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)Z-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene.or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -N(R')C(S)NR'4alkylene or substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')Z-N=N-, -N(R')C(NCN)NR'-(alkylene. or substituted alkylene), -N(R')C(NNO2)NR'-(alk.ylene or substituted alkylene), -N(R')C(NCOOR')NR'-(alkylene or substituted alkylene), and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
R is I=1, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
Ri is H, an aniino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
R2 is OH, an ester protecting group, resin,.at least one amino acid, polypeptide, or polynucleotide;
each R. is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8; and Y independently selected from the group consisting of OR", NR"R", NC(O)R"
where each R" is is independently H, alkyl, substituted alkyl.

Such non-natural amino acids are optionally in the form of a salt, or are incorporated into a non-natural amino acid polypepdde, polymer, polysaccharide, or a polynucleotide and optionally post.translationally modified.
1002741 In addition, the following amino acids are included:

N,O-,_:..~_N,N1k1 NNi N,O^ N=N^ H
H~NH HzNH ~N _ OH H H2N OH
on no o and 0 wherein such compounds are optionally 'amino protected, optionally earboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof,. or are incorporated into a non=natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide~and optionally posttranslationallymodified.
C. Non-Natural Amino Acids Containing an Indolyl Functional Group 1002751 Non-natural amino acids containing an indole group are produced by reaction of either a non-natural amino acid containing a hydrazine with a reagent containing a carbonyl group, or a non-natural ainino acid containing a carbonyl with a reagent containing a hydrazine group. This reaction is called the Fisher indole synthesis. This reaction is traditionally carried out under very harsh condition in the presence of strong acids and /or metal ion accelerators, or uder reflux in organic solvents. Fig. 2 describes the mechanisim of this reaction which involves arylhdrazone intermediate formation between aryllrydrazine and carbonyl compound, followed by [3,3] sigmatropic rearrangement to form the indole product after elimination of ammonia.
100276) In one enibodiment the reactions between carbonyl and arylhydrazines is perfomed in aqueous buffer at room temperature. In another embodiment the reactions between carbonyl and arylhydrazines is perfonied at pH of about 1 to about 6 and about 2 to about 4. In addition,.
the reaction is accelerated, for example, by performing the hydrazone intermediate formation and the rearrangement step to form the indole at different pH. In one embodiment the formation of hydrazone intermediate is realized at pH 5. In another embodiment the rearrangement step is perfomed at pH 1. Fig. 3-6, and 8-11 describe the effect of the pH on Fisher irtdole synthesis.
1002771 In certain:embodiments, metal ions are,used to accelerate_the rate of formation of indole product.
Fig. 12 illustrates non limiting examples of metal ions that have.
accelerating effect on the rate of formation of the indole product. In another embodiment,, the amount of organic solvent in the reaction milieu has an effect on tlie rate of formation of indole product. Fig. 14 describes the effect of the solvent on the rate of formation of the indole product.
1002781 The reagents used in this reaction are optionally further linked to,a desired functionality. In some enibod'nnents, the non-natural amino acid is incorporated into a polypeptide, wheretipon reaction with the appropriate reagent. aconjugate is formed between the polypeptide and molecule of interest.
1002791 Such aniino acids include amino acids having the structure of Formula (XV):

R3 A_ B_1 ]

R i _ R2 H
0 (XV) wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene;_ lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene,.alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alka .rylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO{alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)i`'(R')-, -N(R')C(NCN)N(R')-, -NI(R')C(NNOz)N(R')-, -N(R')C(NCOOR')N(R')-, -N(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')Z-N=N-, and -C(R')2-N(R')-N(R')- and each R' is independently H,. alkyl, or substituted alkyl;

Ra N Re ( ~ )a RS Ra N
R H H
N H Rs N R5 ~ RS RS ~-, I R5 Ra \' ~~ R5 R5 ~ R Ra R5 R6 J is R5 Rs R5 5 or Ri is 1-1, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
each R5 is independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene.oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-0N(R")2, CN, NOz, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted:aryl), -C(O)R", -C(O)2R", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl,.heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, L-Y, or when more than. one R" group is present, two R"
optionally form a heterocycloalkyl;
when more than one R5 group is present, two R5 optionally form a heterocycloalkyl or an aromatic heterocycloalkyl;
n is 0, 1, 2, or 3 and m is 0, 1, 2, or 3, provided that at least one of n or m is not 0;
wherein, each ring in structures 1, 2, 3, and 4 that has an associated R.
group can contain 0, 1, or 2 R.
groups and each R. independently selected from the group cojisisting of H, halogen, alkyl, subsituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)kR", where each R" is independently H, alkyl, or substituted alkyl; or, when more than one R, group is present; two R.
opfionally form an aryl, cycloalkyl or heterocycloalkyl;
Y is selected from: a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore; a metal-containing moiety; a radioactive moiety; a novel fiuictional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-Iinked sugar; a redox-active agent; an amino thioacid; a toxic moiety; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an interealating group; a chromophore;
an energy transfer agent; a biologically active agent (in which case, the biologically active agent can include an agent with therapeutic activity and the non-natural amino acid polypeptide or modified non-natural amino acid can serve either as a co-therapeutic agent with the attached therapeutic agent or as a means for delivery the therapeutic agent to a desired site within an organism); a detectable label; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron-capture agent; a derivative ofbiotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimiotope, a receptor, a reverse micelle, and any combination thereof;
L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or subsfituted alkylene)-, -N(R')C(O)O-, -(alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)-S(O)k-( alkylene or substituted alkylene)-S-, -(alkylene or substituted alkylene)-S-S-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')Z-N=N-, and.-C(R')2-I~'(R')-N(R')-, where k is 1, 2 or 3 and each R' is independently H, alkyl, or substituted allcyl or the -A-B-J groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising an indole portion.
1002801 In one enibodiment, Y is selected from a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group;
at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;

1002811 In a further embodiment is a compound having the structures of compounds 1-4:

Ra H R3 R. R.
N
R RA. N R3 A
3 B R B~/
R. R21 ~ R, ~N RZ n Rs j H R4 0 R5 H~ O 2~ m~ R
s R5 Rs Re N Rj i $
R3 3 A`B/ R5 R3 3 A,H,, R5 RR. ! Rs R N R
m Rs R Rj~ R2 Rt~H R4 O Z 3 e H R40 4 R5 R5 wherein:
A is optional, and wlten present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker, linked at one end to an indole-containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene; lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)Z-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene'or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(.R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNOZ)N(R')-, -N(R')C(NCOOR')N(R')-, -N(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-and each R' is independently H, alkyl, or substituted alkyl;
R, is H, an aniino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
n is 0, 1, 2, or 3, and m is 0, 1, 2, or 3, provided that at least one of n or m is not 0;
wherein, each ring in structures 1, 2, 3, and 4 that has an associated R.
group can contain 0, 1, or 2 R.
groups and each R. is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)LR", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted all.yl; or when niore than one R, group is present, two R, optionally form an aryl, cycloalkyl or heterocycloalkyl;

each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R, or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
each R$ is independently H, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, OH, NH2, CN, NO., -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R", -C(O)2R", or -C(O)N(R )Z, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one RW' group is present, two R" optionally form a heterocycloalkyl;
or R5 is.L-X, where, X is a selected from the group consisting of: a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluoropltore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety;
an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chetniluniinescent group; an electron dense group; a magnetic group; an intercalating group; a chroniophore; an energy transfer agent; a biologically active agent; a detectable label; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron-capture agent; a derivative of biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an abzyme,. an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone;
an antioxidant, an aptamer', a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)t(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -(alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)-S(O)k-( alkylene or substituted alkylene)-S-, -(alkylene or substituted alkylene)-S-S-, -S(O)kN(R')-, -N(R')C(O)N(R')-õ-N(R')C(S)N(R')-, -N(R')S(0)kN(R')-, -N(R')-N=, -C(R')=N-. -C(R')=N-N(R')'.-, -C(R').=N-N=, -C(R')z-N=N-, and -C(R')2=N(R') N(R')-, where each R' is independently.H, alkyl, or substituted-alkyl;
when more than one R5 group is present,. two ortho R$ groups can optionally form aheterocycloalkyl or an aromatic heterocycloalkyl;
or the -B-indole containing moiety together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl coniprising at least one indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable-prodrug or solvate thereof.
1002821 In one emboditnent, X is selected from a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker;at least one amino acid; at least one sugar group;
at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;
1002831 In a further enibodinient is a compound having the structures of compounds 1-4, wherein A is a bond, substituted. or unsubstituted lower alkylene, or an unsubsimted or substituted arylene selected from the group consisting of a phenylene, pyridinylene, pyrimidinylene or thiophenylene. In a. further embodiment is a compound having the structures of compounds 1-4, wherein A is bond. In a further embodiment is a compound having the. structures of compounds 1-4, wherein A is substituted or uns"ubstituted lo.werbeteroalkylene. In a further embodiment is a compound having the structures of comp.ounds 1-4, wherein A is a phenylene. In a further embodiment` is a compound having tlie structures of compounds 1-4, wherein A is a substituted lower heteroalkylene,, whereinthe.subsituent is a single =O group. In a further embodiment is a compound having the structures of compounds 1-4, wherein A is a substituted lower alkylene,.
wherein the substituent is the single =0 group. In yet a fiu-ther embodiment is a compound having the structures of compounds 1-4, wherein B is a bond, lower alkylene, substituted lower alkylene, -O-(alkylene or substituted alkylene)-, -CON(R")-, -NR'-(;alkylene or substituted alkylene)- -N(R")CO-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -CON(R")-(atkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted" alkylene)-, or -S(O)2(alkylene or substituted alkylene)-. In a further embodiment is a compound having the structures of compounds 1-4, wherein B is a bond. In a further embodiment is a cornpound having the structures of compounds 1-4, wherein B is -O(CHZ)-, -NHCH2-, -NHCO-, -C(O)-, -C(O)-(CHZ)-, -CONH-(CH+, -SC112-, -S(=O)CH2-, or -S(O)ZCH,-. In sonte embodiments are compounds having the structures of compounds 1-4, wherein R5 is -OH, -NH2, or NOz. In another embodiment is a compound having the structures of compounds 1-4, wherein Ri is H, tert-butyloxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In a further embodiment is a compound having the structures of compounds 1-4, wherein Ri is a resin,_at least one amino acid, polypeptide, or polynucleotide. In another enibodiinent is a compound having the structures of compounds 1-41 wherein R2 is OH, 0-methyl, 0-ethyl, or O-t-butyl. In a further embodiment is a.compound having the structures of compounds 1-4, wherein R2 is a resin, at least. one amino acid,.polypeptide, or polynucleotide. In yet=a ,further embodiment is a compound having the structures `of compounds 1-4, wherein R2 is a polynucleotide. In ;another embodiment is a compound having the structures of compounds 1-4, wherein R,. is ribonucleic acid (RNA).
In a further embodintent is a compound having the 'stivctures of compounds 1-4, wherein R2 is tRNA. In a further embodiment is a compound having the structures of compounds 1.-0, wherein said tRNA
specifically recognizes a.selector codon.
In a further emboditnent is a compound having the structures of compounds 1-4, wherein said selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon. In a further embodiment is a compound having the structures of compounds 1-4, wherein R2 is a suppressor tRNA. In a further embodiment is a compound having the structures ;of compounds 1-4, wherein X is a biologically active agent selected from the group consisting of a peptide, protein, enzyme, antibody, drug, dye, lipid, nucleosids, oligonucleotide, cell, virus, liposome, microparticle, and .micelle,_ In a further embodiment is a compourid having the structures of compounds 1-4, wherein X is a drug., selected. from the group consisting of an antibiotic, fungicide, anti-viral agent, anti-inflammatory agent; :anti-aumor agent, cardiovascular agent, anti-anxiety agent, hormone, growth factor, and steroidal agent. Iin'a further embodiment is a compound having the .structures of compounds 1-4, wherein X is an enzyme selected from ihe group consisting of horseradish peroxidase; alkaline phosphatase, (3-galactosidase, and glucose.oxidase. In another embodiment is a compound.having the structures of compounds 14, wherein X is a detectable label selected from the group consisting of a fluorescent, phosphorescent, chemiluminescent, chelating, electron dense, magnetic, intercalating, radioactive, chroniophoric, and energy transfermoiety.ln a furtlier embodiment..is a compound having the structures of compounds 1-4, wherein X is a polymer comprising alkyl, substituted alkyl, alkenyt, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylaikoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl. In a further embodiment is a compound having the structures of compounds 1-4, wherein said polymer comprises polyalkylene oxide or substituted polyalkylene oxide. In another enibodiment is a compound having the structures of compounds 1-4, wherein said polymer comprises -[(alkylene or substituted alkylene)-O-( hydrogen, alkyl, or substituted alkyl)],;, wherein x is from 20-10,000. In a further embodiment is a compound having the structures of compounds 1-4, wherein said polymer is m-PEG lsaving a molecular weight ranging from about 2 to about 40 KDa. In certain embodiments, compounds of Formula.(XV) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (XV) are stable for, at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (XV) are stable for at least; 5 days under mildly acidic conditions.
In certain embodiments, =such acidic conditions are pH about 2 to about 8.
1002841 Non=limiting examples oTsuch amino acids include amino acids having the following structures:
O\N NM 1 NH
NH NH F

OMe H=N OH HZN OH H2N OH H-N oM H2N Oli / , ~ \
~ \
Mel7 ~ N
\ NH
I / I ~ ~ I /

HxN Of{ MzN OH
0 , or 0 ; or a salt thereof; or a polypeptide incorporating at any position of the compounds. Such non-natural amino acids are optionally in the form of a salt, or are incorporated into a.

non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.
1002851 ln some embodiments are compounds further having the structures 5-8:
Ra R N H /H Ra a~
t' R5 Rt.~T RZ n H Ro m RS
r Rs Rj. R2 R5 O Rs Ra R H RS
a n ~~ RS RS
m Rg R5 Ri.~T R2~ R5 R Ri~N R2 RS

n is 0, 1, 2, or 3, and m is 0, 1, 2, or 3,, provided that at least one of n or m is not 0;
wherein, each ring in structures 5, 6, 7 and 8 that has an'associated R0 group can contain 0, 1, or 2 R, groups and each R. is, independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")z, -C(O)R", -C(O)N(R")Z, -OR", and -S(O)kR", where k is 1, 2, or 3, where each.R" is independently H, alkyl, or substituted alkyl; or when more than one R. group is present, two R, optionally form an aryl, cycloalkyl or heterocycloalkyl.
1002861 In another embodiment are compounds having the structures 9-12:
(CHz)nX,(CO)yX2(CHz)m~.\ N (CHz)nXt(CO)y~+z(CHz)m~ H Ra Ri`N 4.1R2 9 ~~ / RS Ri, N Rz 10 R/~ ~ ~n +
H O Ra R$ H R4 O m R5 RS
R. Rs RS H
(CH,)õXI(CO),xz(CHz),,, n I NH RS (CH2)oX,(CO)yXz(CHz),õ- N Rs Ri.NR, 11 R. Ra Rs Ri,N, Rz 12 H R~40 RS RS H R4 0 RS Rs wherein n is 0, 1, 2, 3, or 4; m is 0, 1, 2, 3, or 4; provided that n + m is 1, 2, 3, 4, or 5; X, and X2 are independently a bond, 0, or NH; and y is 0 or 1.

1002871 In yet another embodiment are conipounds, selected from the group consisting of:

O O
N N O
HZN OFI H ( / / I{zN ~OH NI

O
OH NOZ
r ' H H
O N~~~N
O O O N
Oli OH ~O ' OH
HzN f' HZN HxN OH H21`
O O O O
1Rv H2N OH H-N Ofl 0 , or 0 or a salt thereof; or a polypeptide incorporating at any position any of the compounds.

C. Cellular Uptake of Non-Natural Amino Acids 1002881 Non-natural amino acid uptake by a eukaryotic cell is one issue that is typically considered when designing and selecting non-natural amino acids, including but not limited to, for incorporation into a protein.
For example, the high charge density of a-amino acids suggests that these conipounds are unlikely to be cell permeable. Natural amino acids are taken up into the eukaryotic cell via a collection of protein-based transport systems. A rapid screen can be done which assesses which non-natural amino acids, if any, are taken up by cells. See, e.g., the toxicity assays in, e.g., the U.S. Patent Publication No. 2004/198637 entitled "Protein Arrays," which is herein incorporated by reference in its entirety, and Liu, D.R. & Schultz, P.G. (1999) Progress.toward the evolution of an organism with an expanded genetic code.
PNAS United States 96:4780-4785. Although uptake is easily analyzed with various assays, an altemative to designing non-natural amino acids that are amenable to cellular uptake pathways is to provide biosynthetic pathways to create amino acids in viva.
1002891 Typically, the non-natural amino acid produced via cellular uptake as described hcrcin is produced in a concentration sufficient for efficient protein biosynthesis, including but not limited to, a natural cellular amount, but not to such a degree as to affect the concentration of the other amino acids or exhaust cellular resources.
Typical concentrations produced in this manner are about 10 mM to about 0.05 mM.

D. Biosyntliesis of Non-Natural Amino Acids 1002901 Many biosynthetic pathways already exist in cells for the production of amino acids and other compounds. While a biosynthetic method for a particular non-natural amino acid may not exist in nature, including but not limited to, in a cell, the methods and compositions described herein provide such methods. For example, biosynthetic pathways for non-natural amino acids are optionally generated in host cell by adding new enzymes or niodifying existing host cell pathways. Additional new enzymes include naturally occurring enzymes or artificially evolved enzymes. For exaniple, the biosyntliesis of p-aminophenylalanine (as presented in an example in WO 2002/085923 entitled "In vivo incorporation of t-nnatural amino acids") relies on the addition of a combination of known enzymes from other organisms. The genes for these enzymes can be introduced into a eukaryotic cell by transforming the cell with a plasmid comprising the genes. The genes, when expressed in the cell, provide an enzymatic pathway to synthesize the desired compound. Examples of the types of enzymes that are optionally added are provided herein. Additional enzymes sequences are found, for exaniple, in Genbank. Artificially evolved enzymes can be added into a cell in the same manner. In this manner, the cellular machinery and resources of a cell are manipulated to produce non-natural amino acids.
1002911 A variety of methods are available for producing novel enzymes for use in biosynthetic pathways or for evolution of existing pathways. For example, recursive recombination, including but not limited to, as developed by Maxygen, Inc. (available on the world wide web at www.rrtaxygen.com), can be used to develop novel enzymes and pathways. See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNA shuffling, Nature 370(4):389-391; and, Sterruner, (1994), DNA shuffling by random frag-nentation and reassembly: hi vitro reconibination for ntolecular evolution, Proc. Natl. Acad. Sci. USA., 91:1 0747-1 075 1. Similarly DesignPathTM, developed by Genencor (available on the world wide web at genencor.com) is optionally used for nietabolic pathway engineering, including but not limited to, to engineer a pathway to create a non-natural amino acid in a cell. This technology reconstructs existing pathways in host organisms using a combination of new genes, including but not limited to, those identified through functional genomics, and molecular evolution and design. Diversa Corporation (available on the world wide web at diversa.com) also provides technology for rapidly screening libraries of genes and gene pathways, including but not limited to, to create new pathways for biosynthetically producing non-natural amino acids.
1002921 Typically, the non-nattual amino acid produced with an engineered biosynthetic pathway as described herein is produced in a concentration sufficient for efficient protein biosynthesis, including but not limited to, a natural cellular amount, but not to such a degree as to affect the concentration of the other aniino acids or exhaust cellular resources. Typical concentrations produced in vivo in this manner are about 10 niM to about 0.05 mM. Once a cell is transformed with a plasmid comprising the genes used to produce enzynies desired for a specific pathway and a non-natural amino acid is generated, in vivo selections are optionally used to further optimize the production of the non-natural amino acid for both ribosomal protein synthesis and cell growth.
E. Additional Syntlietic Methodology 1002931 "Che non-natural amino acids described herein are optionally synthesized using documented methodologies or using the techniques described herein or by a combination thereof. As an aid, the following table provides various starting electrophiles and nucleophiles which are optionally combined to create a desired functional group. The information provided is meant to be illustrative and not limiting to the synthetic tec}miques described herein.

Table 1: Examples of Covalent Linkages and Precursors Thereof Covalent Linka e Pfoduct . J Electio `hile : : . :. . .: Nucleo hile Carboxamides Activated esters amines/anilines Carboxaniides acyl azides amines/anilines Carboxacnides acyl halides amines/anilines Esters acyl halides alcohols/ henots Esters acyl nitriles alcohols/phenols Carboxarnides acyl nitriles amines/anilines Imines Aldehydes amines/anilines Hydrazones aldehydes or ketones Hydrazines Oximes aldeh des or ketones H drox lamines Alk 1 amines alkyl halides amines/anilines Esters alkyl halides carboxylic acids Thioethers alkyl halides Thiols Ethers alkyl halides alcohols! henols 'Thioethers alkyl sulfonates Thiols Esters alkyl sulfonates carboxylic acids Ethers alkyl sulfonates alcohols! henols Esters Anhydrides alcohols/phenols Carboxamides Anhydrides amines/anilines Thiophenols aryl halides Thiols Aryl an-tines aryl halides Amines Thioethers Azindines Thiols Boronate esters Boronates Glycols Carboxamides carboxylic acids amines/anilines Esters carboxylic acids Alcohols h drazines Hydrazides carboxylic acids N-acylureas, or Anhydrides carbodiimides carboxylic acids Esters diazoalkanes carboxylic acids Thioethers Epoxides Thiols Thioethers haloacetamides 'I'hiols Ammotriazines halotriazines amines/anilines Triazinyl ethers halotriazines alcohols/phenols Amidines inudo esters amines/anilines Ureas Isoc anates amines/anilines Urethanes Isocyanates alcohols/phenols Thioureas isothiocyanates aniineslanilittes Thioethers Maleimides Thiols Phosphite esters phosphoramidites Alcohols Silyl ethers silyl halides Alcohols Alkyl amines sulfonate esters amines/anilines Thioethers sulfonate esters Thiols Esters sulfonate esters carboxylic acids Ethers sulfonate esters Alcohols Sulfonamides sulfonyl halides amines/anilines Sulfonate esters sulfon 1'halides phenols/alcohols 1002941 In general, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles, wherein an attacking nucleophile brings an electron pair to the carbon electrophile in order to form a new bond between the nucleophile and the carbon electrophile.
1002951 Non-limiting examples of carbon nucleophiles include, but are not limited to alkyl, alkenyl, aryl and alkynyl Grignard, organolithium, organozinc, alkyl-, alkenyl , aryl- and alkynyl-tin reagents (organostannanes), alkyl-, alkenyl-, aryl- and alkynyl-borane reagents (organoboranes and organoboronates); these carbon nucleophiles have the advantage of being kinetically stable in water or polar organic solvents. Other non-limiting examples of carbon nucleophiles include phosphorus ylids, enol and enolate reagents; these carbon nucleophiles have. the advantage of being relatively easy to generate from precursors. Carbon nucleophiles, when used in conjunction with carbon electrophiles, engender new carbon-carbon bonds between the carbon nucleophile and carbon electrophile.
1002961 Non-limiting examples of non-carbon nucleophiles suitable for coupling to carbon electrophiles include but are not limited to primary and secondary amines, thiols, thiolates, and thioethers, alcohols, alkoxides, azides, semicarbazides, and the like. These non-carbon nucleophiles, when used in conjunction with carbon electrophiles, typically generate heteroatom linkages (C-X-C), wherein X is a hetereoatom, including, but'not limited to, oxygen, sulfu.r, or nitrogen.
1002971 In one embodiment is a method of making a compound of structures *1 or 2 comprising reacting a compound of Formula (11) with a carbonyl-containing compound, wherein the compound of Formula (li) is, Ra Ri N Ra H' N.R3 Ra 0 R, (11) wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene; or substituted aralkylene;
B is optional, and when present is a Iinker, linked at one end to an indole containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)z-, -OS(O)Z-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylenc or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, =N-O-(alkylene or substituted alkylene), -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')Z-N=N-, and -C(R')2-N(R')-N(R')-;,and each R' is independently H, alkyl, or substituted alkyl;
R' is Fl, an aniino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and RZ is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
each R. is independently selected from the group consisting. of H,:halogen, alkyl; substituted alkyl, CN, NO2, -N(R')2, -C(O)R', -C(O)N(R')Z, -OR', and -S(O)kR', where k is 1, 2 or 3 and each R' is independently H, alkyl, or substituted alkyl;
R3 and R4 are independently hydrogen or amine protecting group, including, but not limited to, i -3-1 y0 OCC13 ,Sf\ /Q~ ~ ~ =C
1Q( Q I0l NQ2 CZHS

yQ_~<CCH =G CN Y' ~=C~ H3 or ~rQ~/~SiMc3 0 O H3C 3, + Q CH7 O

1002981 In a further embodiment is a method of making a compound of structures 1 or 2, wherein A is substituted or unsubstittited lower alkylene, or an unsubsituted or substituted arylene selected from the group consisting of a phenylene, pyridinylene, pyrimidinylene or thiophenylene. In another embodiment is a method of making a compound of structures 1 or 2, wherein A is substituted or unsubstituted lower heteroalkylene.
1002991 In a further embodiment is a method of making a compound of structures I or 2, wherein A is a substituted lower heteroalkylene, wherein the subsituent is a single =0 group.
In yet a further embodiment is a method of making a compound of structures 1 or 2, wherein A is a substituted lower alkylene, wherein the substituent is the single =0 group.
1003001 In a further embodiment is a method of making a compound of structures 1 or 2, wherein B is lower alkylene, substituted lower alkylene, -0-(alkylene or substituted alkylene)-, -CON(R")-, -NR"-(alkylene or substituted'alkylene)-, -N(R")CO-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -S(atkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-, or -S(0)2(alkylene or substituted alkylene)-; wherein each R" is independently H, alkyl, or substituted alkyl.
1003011 In a further embodiment is a method of making a compound of structures l or 2, wherein B is -O(CH2)-, -NHCH2-, -NHCO-, -C(O)-, -C(O)-(CH2)-, -CONH-(CHZ)-, =SCHz-, -S(-0)CH2-, or -S(0)2CH2-.
1003021 In a further embodiment is a method of making a compound of structures I or 2, wherein Ri is H, tert-butyloxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz).
1003031 In a further embodiment is a method of niaking a compound of structures I or 2, wherein R3 and R. are ~
iCH, /CN _ C H
hydrogen, CCH3, =C,CN, ~ 0 , or C2H5.
1003041 In a further embodiment is a method of making a compound of structures 1 or 2, wherein Ri is a resin, at least one amino acid, polypeptide, or polynucleotide. In another embodiment is a method of making a compound of structures I or 2, wherein R, is OH, 0-methyl, O-ethyl, or O-t-butyl. In-yet a further embodiment is a method of making a compound ofstructures. l or 2, wherein RZ is a resin, at least one amino acid, polypeptide, or polynucleotide.
1003051 In some embodiments are methods of making a compound of structures 1 or'2, wherein the compound of Formula (II) has the structure:
R.
(CH2)nXI(C0)yX2(CH2)m-B ~
I ~
RI~N ~ R2 13 R N' 0 ~

wherein, n is 0, 1, 2, 3, or 4; m is 0, 1, 2, 3, or 4; provided that n+ m is 1, 2, 3, 4, or 5; XI and X, are independently a bond, 0, or NH; and y is 0 or 1.
1003061 In a further embodiment is a method of making a.compound of structures I or 2, wherein R2 is a polynucleotide. In another embodiment is a method of making a compound of structures 1 or 2, wherein R, is ribonucleic acid (RNA). In a further embodiment is a method of making a compound of structures 1 or 2, whereinR2 is tRNA. In a further embodiment is a method of making.a compound of structures I or 2, wherein said tRNA specifically recognizes a selector codon. In yet a further enibodiment is a method of making a compound of structures I or 2, wherein said selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon. In a further embodiment is a method of making a compound of structures 1 or 2; wherein R~ is a suppressor tRNA.
1003071 In afurther embodiment is a method of making a compound of structures 1 or 2, selected from the group consisting of H
HN N, NH:

NHz \-~\
H ~ NH
H N OH HiN 1't Hz,~ 2 ~ O 0 O~~~y~ O~/~y \
H N H H N H A OH H I/ N'~Y
Z H , or Z H I

1003081 In sonie embodiments are niethods of making a compound of structures I
or 2, corresponding to Formula (IV):
Ra H
Ra N~ ,Rd I I
Rj H
Rt,, Ra 0 (IV) wherein, each R, is independently selected from the gtoup consisting-of H, halogen, alkyl, substituted alkyl, CN, NO2, -N(R")2, -C(O)R", -C(O)N(R")Z, -OR", and -S(O)kR", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl.

1003091 In a further embodiment is a method of making a conipound of structures 1 or 2, selected from the group consisting of:

N
~ H
NHZ N"N~
( }~ ( ~ \ \
OH OII OH
HaN -H3N H2N

GN
N~y~
\ ~ \ I N/ CN
OH OH
HZN HzN
o ;or 1003101 In a further enibodiment is a method of making a compound of structures 1 or 2, further contprising reacting a compound of Formula (V) with a hydrazine containing agent; wherein the conipound of Formula (V) is:

R A \B1-1 jR
R, R:

H EQ (v), wherein:
A is optional, and when present is lower alkylene,.substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenytene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heteroeycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -0-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, where..k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, and -N(R')CO-(alkylene or substituted alkylene)-, where each R' is independently H, alkyl, or substituted alkyl;

0 R' 0 R,' Rõ SR" R~~ +N
0 S O o ORn N
y,,/~r'" ,s~~ \ 0 /
`~, Jis O
Rõ R" Rõ Rõ
s . s b or R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
each R" is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R" optionally form a heterocycloalkyl;
R, is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl goup, including a carbonyl group, protected carbonyl group, including a protected carbonyl group, or masked carbonyl group, including a masked carbonyl group;
or the -J-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a carbonyl group, protected carbonyl group, including a protected carbonyl group, or masked carbonyl group, including a masked carbonyl group;
with a proviso that when A is phenylene and each R3 is H, B is present; and that when A is -(CH2)4- and each Rj.is H, B is not -NHC(O)(CHZCH2)-; and that when A and B are absent and each R3 is H, R is not methyl.
1003111 In a further.embodiment is a method of making a compound of structures 1 or 2, corresponding to Formula (VI):

~R
Rj~N H
;RB2 0 (VI) 1003121 In a further embodiment is a method of making a compound of structures 1 or 2, wherein A is substituted or unsubstituted lower alkylene, or an unsubsituted or substituted arylene selected from the group consisting of a plienylene, pyridinylene, pyrimidinylene or method.
1003131 In a further embodiment is a method of making a compound of structtues I or 2, wherein B is lower alkylene, substituted lower alkylene, -O-(alkylene or substituted alkylene)-, -CON(R")-, -NR".-(alkylene or substituted alkylene)-, -N(R")CO-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-, or -S(O)2(alkylene or substituted alkylene)-. In a further embodiment is a method of making a compound of structures ] or 2, wherein B is -O(CH2)-, -NHCH2-, -NHCO-, -C(O)-, -C(O)-(CH2)-, -CONH-(CHZ)-, -SCH2-, -S(=O)CH2-, or -S(O)2CH2-.

1003141 In a further embodiment is a inethod of making a compound of structures 1 or 2, selected from the group consisting of:

0 fi ~ ~
~
H
H,N Oil H1 j N OI{ HN H H NH H OH
a HZN H2N H7N
O
O O
O H^t ~ FtN'~O
F1zN OH HN Oil H,N OH H2N OFi o , , , or 1003151 In a further embodiment is a method of making a compound.of structures I or 2, corresponding to Formula (VII):
Ra R, By R

R.
R.
RI~N RZ
H
0 (VII), wherein, each R. is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R"),, -C(O)R", -C(O)N(R")2, -OR", and -S(O)kR", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl.
(00316) In a further embodiment is a niethod of making a compound of structures 1 or 2, selected from the group consisting of:
o oa cl o, ~ ~;~ ~~
`~~ ~/ ~' On Hc~ H>N ~OH .~ OH 14,N OH
O O O
e > , , 0'I O

10 ~ O~/'`. \
I/ I, o^ y H0 ~/
H-N Oil oti 0 I,hN H
O Q HZN COOH
> , > >
~ ~ i OA
N-y ' / I /
H; H,N OH H2N H H=N OH
, or (00317) In a further embodiment is a method of making a cornpound of structures l or 2, corresponding to Formula (VIII):
Ra BYz Y
R, R.
R,, N R.Z
H
O
wherein, each R. is independently selected from.the group consistingof H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R",--C(O)N(R")2, -OR";.and -S(O)kR", where.k is 1, 2, or 3, where each R" is independently H, alkylõor substituted alkyl;
Y and Z are independently selected from the group consisting of=OI-I, alkyl substituted oxygen, -SH or alkyl substituted sulfur and when Y and Z taken together can form a cycloalkyl ring.
1003181 In a further embodiment is a method of making a compound of structures 1 or 2, selected from the group consisting of:

HzA tiN }t ttN H oH
H
O
H
\ \ \ ~/ ~ \

H y H H,N H H2N tl tizN H

H ~ O Qj \ `S~ \ '~ i ~~/~

/ \ HHz h H [1z Oli H2N H HxN Oti r O
O>
O\
iO
H
Hz1 ~OH H,1\T
O or 1003191 In a further embodiment is a method of making a conipound of structures I or 2, corresponding to Formula (IX):
R, l3vN, ~.
RI
a RF

Ri~ N H
0 (IX) wherein, each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")Z, -OR", and -S(O)kR", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl;
Y is independently selected from the group consisting of OR", NR"R", NC(O)R"
where each R" is independently H, alkyl, or substituted alkyl.
1003201 In=a further embodiment is a method of making a compound of structures 1 or 2, selected from the grotip consisting, o f:

.?J,N~
~ .1 I ~ .Nbn I ~ H
i i HzN pH H2N H2N H
0 Oi N' O

O
1003211 In a fui-ther enibodiment is a method of making a compound of structures 1 or 2, corresponding to Formula (X):

R' O
R, R, I
Ri, N RZ R~

H 0 (X) wherein, each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")Z, -OR", and -S(O)kR", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl; or when more than one R. group is present, two R.
optionally form a cycloalkyl or heterocycloalkyl;
R3. and R4 are independently H, halogen, CN, NO2, alkyl, substitutcd alkyl, N(R')z, C(O)R', -C(O)N(R');; -OR', and -S(O)kR', where k is 1, 2, or 3, where each R' is independently H, alkyl, or substituted alkyl;
X is.C, N, or S, with the proviso that when X is 0 or S, Ra cannot be H, halogen, CI`?, NOZ, alkyl, .substituted alkyl, N(R')2, C(O)R', -C(O)N(R')Z, -OR', and -S(O)kR'; where k is 1, 2, or 3, and n is 0, lor2.

1003221 In a fitrther embodiment is a method of making a compound of structures I or 2, selected from the group consisting of:
OMe $jo 0 H2N H HZN OH HzN OH F H2\ OH HaN OFI

0 Q O , 0 , 0 MeO O Me0 O

/ F
\ I \ ( \ I O
H=N -ly OH H2N OH H'N OH 112N OH
O 0 O Or 0 1003231 In yet a further embodiment is a method of niaking a compound of structures 1 or 2, corresponding to Formula (XI):
z ~CRa)n\ / ~~, B
RI\N R2 H
0 (Xl) wherein, each Rais independently selected from the group consisting of H, halogen, alkyl, substituted all.yl;
Y and Z are independently selected from the group consisting of -(OH, alkyl substituted oxygen, =.SI-I 'or alkyl substituted sulfur and when Y and Z taken together canform a.cycloalkyl ring.
1003241 In a further embodiment is a method of making a compound of structures 1 or 2, selected from the group consisting of:

^
po O
~ ~ O O~ N}i Jl O
NH Nll NH

HZV 11 Hzn~fi HZN~OH H=N~O HzN ~OH 112N OH
a 0 , 0 0 0 iO
s H2N UH H,N OH tl,N OH HIN fl l1=N H H2N Oli I
o 0 0\

H
fIZNOH H2N
or 1003251 In a ftirther embodiment is a method of making a comp.ound of structures I or 2, corresponding.to Formula (XII):

B -r N_ Y
(CR ) n R

R~~ Rz O

(XII) wherein, each R. is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2. -C(O)R", -C(O)N(R"),, -OR", and -S(O)kR", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl;
wherein B further comprises -CH=N-O-(alkylene or substituted alkylene)-;
n is 1, 2, or 3; and Y is independently selected from the group consisting of OR", NR"R", NC(O)R"
where each R" is independently H, alkyl,,substituted alkyl.
1003261 In a furthcr embodiment is a.method of making. a conipoundof structures 1 or 2, selected from the group consisting of:

~1'Ni, i;J~ iN.N
I
"2N OH N,NH H,N~ H2N Oll 0 0 or O
1003271 In a further embodiment is a method of making a compound of structures 1 or 2, wherein the compound is reactive with a carbonyl cotitaining agent in aqueous solution under mild conditions.
1003281 In a further embodiment is a method of making a compound of structures 1 or 2, wherein the reaction of the compotmd with the carbonyl containing or protected carbonyl containing agent has at least one of the following characteristics: (i) occurs in a pH range of about 1 to about 6, (ii) generates an indole linkage which is stable under biological conditions; (iii) is site-specific; (iv) does not irreversibly destroy the tertiary structure of a polypeptide; (v) occurs at room termperature; (vi) occurs readily in aqueous conditions; or (vii) is regioselective and/or regiospecific.
I003291 In yet a further embodiment is a method of making a compound of structures I or 2, wherein the mild conditions are pH about I to about 6. In another embodiment is a ntethod of making a compound of structures I
or 2, wherein the mild conditions are pH about 3 to about 6. In a further embodiment is a method of making a compound of structures 1 or 2, the reaction is in an aqueous solution iunder mild conditions.
1003301 In a further embodiment is a method of making a compound of structures 1 or 2, wherein reacting a compound of Formula (V) with a hydrazine containing agent has at least one of the following characteristics: (i) occurs in a pH range of about I to about 6, (ii) generates an indole linkage which is stable under biological conditions; (iii) is site-specific; (iv) does not irreversibly destroy the tertiary structure of a polypeptide; (v) occurs rapidly at room termperature; (vi) occurs readily is aqueous conditions; or (vii) is regioselective and/or regiospecific.

VI. Polypeptides with Non-natural Amino Acids 1003311 For convenience, the form, properties and other characteristics of the compounds described in this section have been described generically and/or with specific examples.
However, die forni, properties and other characteristics described in this section should not be limited to just the generic descriptions or specific example provided in this section, but rather the form, properties and other characteristics described in this section apply equally well to all compounds that fall within the scope of Formulas I-XV, and compounds having the structures 1-4, including. any sub-forntulas or specific compounds that fall within the scope of Formulas I-XV and conipounds having the structures 1-4 that are described in the specification, claims and figures herein.
[003321 The compositions and methods described herein provide for the incorporation of at least one non-natural aniino acid into a polypeptide. The non-natural aniino acid are optionally present at any location on the polypeptide, including any terminal position or any internal position of the polypeptide. Preferably, the non-natural amino acid does not destroy the activity and/or the tertiary structure of the potypeptide relative to the honiologous naturally-occurring amino acid polypeptide, unless such destruction of the activity and/or tertiary stntcture was one of the purposes of incorporating the non-natural amino acid into the polypeptide. Further, the incorporation of the non-natural amino acid into thepolypcptide optionally modifies to some extent the activity (e.g., manipulating the therapeutic effectiveness of the polypeptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics,. pharmacologics and/or pharmacodynamics of the polypeptide (e.g., increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time), providing additional functionality to the polypeptide, incorporating a tag, label or detectable signal into the polypeptide, easing the isolation properties of the polypepfide, and any contbination of the aforementioned modifications) and/or tertiary structure of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide without fully causing destruction of the activity and/or tertiary structure. Such modifications of the activity and/or tertiary structure are often one of the goals of effecting such incorporations, although the incorporation of the non-natural aniino acid into the polypeptide optionally has little effect on the activity and/or tertiary structure of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide.
Correspondingly, non-natural amino acid polypeptides, conipositions comprising non-natural amino acid polypeptides, methods for making such polypeptides and polypeptide compositions, methods for purifying, isolating, and characterizing such polypeptides and polypeptide compositions, and methods for using such polypeptides and polypeptide compositions are considered within the scope of the present disclosure. Further, the non-natural amino acid polypeptides described herein are optionally ligated to another polypeptide (including, by way of exaniple, a non-natural amino acid polypeptide or a naturally-occurring amino acid polypeptide).
1003331 The polypeptide is selected, for example, froni any known therapeutic protein, that is a protein which is known to have a therapeutic effect on a person having a disease, disorder or condition. By way of example only, the polypeptide is selected froin alpha-I antitrypsin, angiostatin, antihemolytic factor, antibody, antibody fragment, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atria] peptide, C-X-C
chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b; gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4; MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3; monocyte inflammatory protein-1 alpha, nionocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF), contplement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidernial growth factor (EGF), epithelial neutrophil activating peptide, crythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), 6brinogen, fibronectin, four-helical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-I, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-garnma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic proteiii, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, soniatostatin, somatotropin, streptokinase, superaritigens, staphylococcal enterotoxin, SEA, SEB, SECI, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNFR), urotensin, VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone receptor.
1003341 The non-natural amino acid polypeptides described herein are optionally produced biosynthetically or non-biosynthetically. By biosynthetically is meant any method utilizing a translation system (cellular or non-cellular), including use of at least one of the following components: a polynucleotide, a codon, a tRNA, and a ribosome. By non-biosynthetically is meant any method not utilizing a translation system: this approach can be further divided into methods utilizing solid state peptide synthetic methods, solid phase peptide synthetic methods, methods that utilize at least one enzyme, and methods that do not utilize at least one enzyme; in addition any of these sub-divisions may overlap with another sub-division and many methods optionally utilize a combination of these sub-divisions.
1003351 The methods, compositions, strategies and techniques described herein are not limited to a particular type, class or family of polypeptides or proteins. Indeed, the scope of the compositions described herein allows for virtually any polypeptide to include at least one non-natural amino acids described herein. By way of example only, the polypeptide is homologous to a therapeutic protein. In a related or further embodiment, the non-natural ainino acid polypeptide is homologous to any polypeptide member of the growth honnone supergene family.
1003361 The non-natural amino acid polypeptides are optionally further modified as described elsewhere in this disclosure or the non-natural amino acid polypeptide is optionally used without further modification.
Incorporation of a non-natural amino acid into a polypeptide is done for a variety of purposes, including but not liniited to, tailoring changes in protein structure and/or function, changing size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, accessibility of protease target sites, targeting to a moiety (including but not limited to, for a polypeptide array), etc. Polypeptides that include a non-natural amino acid can have enhanced or even entirely new catalytic or biophysical properties. By way of example only, the following properties can be modified by inclusion of a non-natural amino acid into a polypeptide:
toxicity,. biodistribution, structwal properties, spectroscopic properties, chen-iical and/or photochemical properties, catalytic ability, half-life (including but not limited to, serum half-life), ability to react with other molecules, including but not limited to, covalently or noncovalently, and the like. Compositions with polypeptides that include at least one non-natural aniino acid are useful for, including but not linuted to, novel tlierapeutics, diagnostics, catalytic enzymes, industrial enzymes, binding proteins (including but not limited to, antibodies), and research including, but not limited to, the study of protein structure and function. See, e.g., Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology, 4:645-652.
1003371 Further, the sidechain of the non-natural amino acid component(s) of a polypeptide provides a wide range of additional functionality to the polypeptide; by way of example only, and not as a limitation, the sidechain of the non-natural amino acid portion of a polypeptide includes any desired functionality.
1003381 In one aspect, a composition includes at least one polypeptide with at least one, including but not liniited to, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more non-natural amino acids. Such non-natural amino acids are optionally the same or different. In addition, there is optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different sites in the polypeptide which comprise 1, 2, 3, 4, 5, 6, 7, 8; 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more different, or the same, non-natural amino acids. In another aspect, a composition includes a polypeptide with at least one, but fewer than all, of a particular amino acid present in the polypeptide is substituted with a non-natural amino acid(s). For a given polypeptide with more than one non-natural amino acids, the non-natural amino acids can be identical or different (such as; by way of exaniple only, the polypeptide can include two or more different types of non-natural amino acids, or can include two of the same non-natural amino acid). For a given polypeptide with more than two non-natural amino acids, the non-natural amino acids can be the same, different or a combination of a multiple number of non-natural amino acids of the same kind with at least one different non-natural amino acid.
1003391 Although embodiments of the non-natural amino acid polypeptides described herein are optionally chemically synthesized via solid phase peptide synthesis methods (such as, by way of example only, on a solid resin), by solution phase peptide synthesis niethods, and/or without the aid of enzymes, other embodiments of the non-natural ainino acid polypeptides described herein allow synthesis via a cell membrane, cellular extract, or lysate system or via an in vivo system, such as, by way of example only, using the cellular machinery of a prokaryotic or eukaryotic cell. In fiuther or additional embodinlents, one of the key features of the non-natural amino acid polypeptides described herein is that they are synthesized utilizing ribosomes. In further or additional embodiments of the non-natural amino acid polypeptides described herein are, the non-natural amino acid polypeptides are synthesized by a combination.of the methods including, but.not limited to, a combination of solid resins, without the aid of eiizynies, via the aid of ribosomes, and/or via an in vivo system.
1003401 Synthesis of non-natural amino acid polypeptides via ribosomes, and/or an in vivo system has distinct advantages and characteristic from a non-natural amino acid polypeptide synthesized on a solid resin or without the aid of enzymes. These advantages or characteristics include different impurity profiles: a system utilizing ribosomes and/or an in vivo system will have impurities stemming from the biological system utilized, including host cell proteins, membrane portions, and lipids, whereas the impurity profile from a system utilizing a solid resin and/or without the aid of enzymes may include organic solvents, protecting groups, resin materials, coupling reagents and other chemicals used in the synthetic procedures. In addition, the isotopic pattern of the non-natural amino acid polypeptide synthesized via the use of ribosomes and/or an in vivo system may mirror the isotopic pattern of the feedstock utilized for the cells; on, the other hand, the isotopic. pattern of the non-natural amino acid polypeptide synthesized on a solid resin. and/or without the aid of enzymes may mirror the isotopic pattern of the amino acids utilized in the synthesis. Further, the non-natural amino acid synthesized via the use of ribosomes and/or an in vivo system may be substantially free of the D-isomers of the amino acids and/or may be able to readily incorporate intetnal cysteine amirio acids into the'stxucture of the polypeptide, and/or may rarely provide,intemal anuno acid deletion polypeptides. On the other hand, a non-natural amino acid polypeptide synthesized via a solid resin and/or without.the use of enzymes may have a higher content of D-isomers of the amino acids and/or a lower content of internal cysteine amino acids and/or a higher percentage of internal amino acid deletion polypeptides. Furthermore, one will be able to differentiate a non-natural amino acid polypeptide synthesized by use of a ribosome and/or an in vivo system from a non-natural amino acid polypeptide synthesized via a solid resin and/or without the use of enzymes.
VIL Con:positions and Methods Conrprising Nucleic Acids and Oligonucleotides A. General Recontbinant Nucleic Acid Methods For Use Herein 1003411 In numerous embodiments of the methods and compositions described herein, nucleic acids encoding a polypeptide of interest (including by way of example a GH polypeptide) will be isolated, cloned and often altered using recombinant methods. Such embodiments are used, including but not limited to, for protein expression or during the generation of variants, derivatives, expression cassettes, or other sequences derived from a polypeptide. In some embodiments, the sequences encoding the polypeptides are operably linked to a heterologous promoter.
[00342) Also described herein are cells that can producenon-natural amino acid polypeptides wherein at least one non-natural amino acid on-the polypeptide comprises a side-chain having a carbonyl, a hydrazine; an indole linkage.. Such cells produce such non-natural amino acid polypeptides using the methods described herein or variants thereof, but biosynthetically produce at least one non-natural amino.
Cells that biosynthesize at least one non-natural amino acid may be produced using the techniques, methods, compositions and strategies described herein or variants thereof 1003431 A nucleotide sequence encoding a polypeptide comprising a non-natural amino acid may be synthesized on the basis of the amino acid sequence of the parent polypeptide, and then changing the nucleotide sequence so as to effect introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of the relevant amino acid residue(s). The nucleotide sequence may be conveniently modified by site-directed mutagenesis in accordance witb documented niethodologies. Altematively, the nucleotide sequence may be prepared by chemical synthesis, including but not limited to, by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced.
For example, several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and assembled by PCR, ligation or ligation chain reaction. See, e.g., Barany, ec al., Proc. Natl. Acad. Sci. 88.
189-193 (1991); U.S. 6,521,427 which are incorporated by reference herein for disclosure of the aforementioned.

(00344) The non-natural amino acid methods and compositions described herein utilize techniques in the field of reconibinant genetics. Basic texts disclosing the general methods of use for the non-natural amino acid methods and compositions described herein include Sanibrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
1003451 General texts which describe molecular biological techniques include Berger and Kinimel, Guide to ,v volume 152 Academic Press, Inc., San Diego, CA
Molecular Clonint; Techniques Methods in Enzymolop (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Ed.).
Vol. 1 or 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook") and Current Protocols in Molecular Bioloev, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) ( Ausubel")).
These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics related to, including but not limited to, the generation of or polynucleotides which include selector codoii.s for production of proteins that include non-natural amino acids, orthogonal tRNAs, orthogonal synthetases, and pairs thereof.
1003461 Various types of mutagenesis are used in the non-natural amino acid methods and compositions described herein for a variety of purposes, including but not limited to, to produce novel synthetases or tRNAs, to mutate tRNA molecules, to mutate polynucleotides encoding synthetases, libraries of tRNAs, to produce libraries of synthetases, to produce selector codons, to insert selector codons that encode non-natural amino acids in a protein or polypeptide of interest. They include but are not limited to site-directed mutagenesis, random point mutagenesis, homologous reconibination, DNA shuffling or other recursive mutagenesis methods, chimeric construction, mutagenesis using uracil containing, templates, oligonucleotide-directed niutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA
or the like, or any combination thereof. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient liost strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis., double-strand break repair, and the like. Mutagenesis, including but not limited to, involving chilneric constructs, are also included in the non-natural amino acid methods and compositions described herein. In one embodiment, niutagenesis can be guided by documented information of the naturally occurring molecule or altered,or mutated naturally occurring molecule, including but not liniited to, sequence comparisons, physical properties, crystal structure or the like.
1003471 The texts and examples found herein describe these and other relevant procedures. Additional information is found in the following publications and references cited.within: Ling et al., Approaches to DNA
ntutagenesis: an overview, Anal Biochem. 254(2): 157-178 (1997); Dale et al., Oligonucleotide-directed random n:utagenesis using the phosphorothioate method, Methods Mol. Biol.
57:369-374 (1996); Smith, In vitro mutagenesis, Ann. Rev. Genet. 19:423-462(1985); Botstein & Shortle, Strategies and applications of in vitro mutagenesis, Science 229:1193-1201(1985); Carter, Site-directed mtrtagenesis, Biochem. J. 237:1-7 (1986); Kunkel, The efficiency of oligomtcleotide directed mutagenesis, in Nucleic Acids & Molecular Bioloav (Eckstein, F. and Lilley, D.M.J. eds., Springer Verlag, Berlin)) (1987);
Kunkel, Rapid and efficient site-specific tnutagenesis without phenotypic selection, Proc. Natl. Acad. Sci, USA 82:488-492 (1985); Kunkel et al., Rapid and efficient site-specific inutagenesis without phenotypic selection, Methods in EnzMiol. 154, 367-382 (1987);
Bass et al., Mutant Trp repressors with new DNA-binding spe.cifrci.ties, Science 242:240-245 (1988); Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith, Oligonucleotirle-directed niulagenesis using M73-der.ived vectors: an efficient and general procedure for the production of point mutations in any DNA fragntent, Nucleic Acids Res. 10:6487-6500 (1982); Zoller & Snuth, Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors, Methods in Enzyniol. 100:468-500 (1983);
Zoller & Smith, Oligonucleotide-directed mutagenesis: a simple metliod using two oligonucleotide primers and a single-stranded DNA template, Methods in Enz ny iol. 154:329-350 (1987);
Taylor et al., The use of phosphorothioate-modified DNA in t-estriction enzyme reactiatis to prepare nicked DNA, Nucl. Acids Res. 13:
8749-8764 (1985); Taylor et al., The rapid generation of oligonucleotide-directed nnitations at high freguency using phosphorothioate-modified DNA, Nucl. Acids Res. 13: 8765-8785 (1985);
Nakamaye & Eckstein, Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis, Nucl. Acids Res. 14: 9679-9698 (1986);
Sayers et al., 5'-3' Exonucleases in phosphorothioate-based oligonucleotide-directed mutrtgenesis, Nucl. Acids Res. 16:791-802 (1988); Sayers et al., Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide, (1988) Nucl. Acids Res. 16: 803-814;
Kramer et al., The gapped duplex DNA approach to oligottucleotide-directed mutatiott construction, Nucl. Acids Res. 12: 9441-9456 (1984);
Kramer & Fritz Oligonucleotide-directed construction of mutations via gapped duplex DN.4, Methods in Enzymol. 154:350-367 (1987); Kramer et al., Intproved enzymatic in vitro reactions in the gapped duplex DNA
approach to oligonucleotide-directed construction of ntutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al., Oligottucleotide-directed construction of trttttations: a gapped duplex DNA
procedure witliout enzymatic reactions in vitro, Nucl. Acids Res. 16: 6987-6999 (1988); Kramer et al., Point Mistnatch Repair, Cell 38:879-887 (1984); Carter et al., Improved oligonucleotide site-directed mutagenesis using M13 vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Intproved oligonucleotide-directed mutagenesis using M13 vectors, Methods in Enzymot. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use of oligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115 (1986); Wells et al., Importance of liydrogen-bonrl formation in stabilizing the transition state of subtilisin, Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloning of a gene codingfor the ribonuclease S proteiii, Science 223: 1299-1301 (1984); Sakmar and Khorana, Total s_vnthesi.s and expression of a gene for the alpha-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells et al., Cassette mutagenesis: an efficient method for generation of tnultiple mtdations at defined sites, Gene 34:315-323 (1985);
Grundstrom et al., Oligonucleotide-directed mutagenesis by inicroscale 'shot-gun' gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki, Oligonucleotide-directed double-strand break repair in plasntids of Escherichia coli: a method for site-specific rnutagenesis, Proc. Natl. Acad.
Sci. USA, 83:7177-7181 (1986);
Arnold, Protein e gineering for unusual environments, Current Oainion in Biotechnology 4:450-455 (1993);
Sieber, et al., Nattire.Biotechnology, 19:456-460 (2001). W. P. C. Stemmer, Nature 370, 389-91 (1994); and, I.
A. Lorimer, 1. Pastan, Nucleic Acids Res. 23, 3067-8 (1995). Additional details on niany of such methods can be found in Methods in Enzymology Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.
1003481 The niethods and compositions described herein also include use of eukaryotic host cells, non-eukaryotic host cells, and organisms for the in vivo incorporation of a non-natural amino acid via orthogonal tRNA/RS pairs. Host cells are genetically engineered (including but not limited to, transformed, transduced or transfected) with the polynucleotides corresponding to the polypeptides described herein or constructs which include a polynucleotide conesponding to the polypeptides described herein, including but not limited to, a vector corresponding to the polypeptides described herein, which can be, for example, a cloning vector or an expression vector. For example, the coding regions for the orthogonal tRNA, the orthogonal tRNA synthetase, and the protein to be derivati2ed are operably linked to gene expression control elements that are functional in the desired host cell. The vector can be, for example, in the form of a plasmid, cosmid, a phage, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide. The vectors are introduced into cells and/or microorganisms by methods including electroporation (Fromm et al., Proc: Natl.
Acad. Sci. USA 82, 5824 (1985)), infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)), and/or the like.
1003491 The engineered host cells can be cultured in conventional nutrient media modified as appropriate for such activities as, for example, screening steps, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic organisms. Other useful references, including but not limited to for cell isolation and culture (e.g., for subsequent nucleic acid isolation) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Techniaue, third edition, Wiley- Liss, New York and the references cited therein;
Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY;
Ganiborg and Phillips (eds) (1995) Plant Cell Tissue and Organ Culture;
Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.
1003501 Several methods of introducing target nucleic acids into cells are available, any of which can be used in methods and compositions described herein. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bonibardment,. and infection with viral vectors (discussed further, herein), etc. Bacterial cells can be used to amplify the number of plasn-tids containing DNA
constructs corresponding to the polypeptides described herein. The bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of inethods (see, for instance, Sambrook). In addition, a plethora of kits are commercially available for the purification of plasmids from bacteria, (see, e.g., EasyPrepT"t, FlexiPrepTM, both from Pharmacia Biotech; StrataCleanT"', from Stratagene;
and, QlAprepTM from Qiagen). The isolated and purified plasmids are then further manipulated to produce other plasniids, used to transfect cells or incorporated into related vectors to infect organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (including but not limited to, shuttle vcctors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Gillam & Smith, Gene 8:81 (1979);
Roberts, et al., Nature, 328:731 (1987);
Schneider, E., et al., Protein Expr. Purif. 6(l):10-14 (1995); Ausubel, Sambrook, Berger (all supra). A catalogue of bacteria and bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of bacteria and bacteriophage (1992) Ghema et al. (eds) published by the ATCC.
Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY. In addition, essentially any nucleic acid (and virtually any labeled nucleic acid, whether standard or non-standard) can be custom or standard ordered from any of a variety of commercial sources, such as the Midland Certified Reagent Company (Midland, TX mcrc.com), The Great American Gene Company (Ramona, CA
available on the World Wide Web at genco.com), ExpressGen Inc. (Chicago, IL available on the World Wide. Web at expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others.

B. Selector Codons 1003511 Selector codons encompassed within the niethods and compositions described herein expand the genetic codon framework of protein biosynthetic machinery. For example, a selector codon includes, but is not limited to, a unique three base codon, a nonsense codon, such as a stop codon, including but not limited to, an amber codon (UAG), or an opal codon (UGA), a unnatural codon, a four or more base codon, a rare codon, or the like. There is a wide range in the number of selector codons that can be introduced into a desired gene or polynucleotide, including but not limited to, one or more, two or more, tluee or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotide encoding at least a portion of a polypeptide of interest.
1003521 In one enibodinient, the methods involve the use of a selector codon that is a stop codon for the incorporation of one or more non-natural amino acids in vivo. For exaniple, an O-tRNA is produced that recognizes the stop codon, including but not limited to, UAG, and is aminoacylated by an O-RS with a desired non-natural amino acid. This O-tRNA is not recognized by the naturally occurring host's aminoacyl-tRNA
synthetases. Site-directed mutagenesis can be used to introduce the stop codon, including but not limited to, UAG, at the site of interest in a polypeptide of interest. See. e.g.. Sayers, J.R., et al. (1988), 5.3' Exonuclease in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 16(3):791-802. When the 0-RS, 0-tRNA and the nucleic acid that encodes the polypeptide of interest are.
combined in vivo, the non-natural amino acid is incorporated in response to the UAG codon to give a polypeptide containing the non-natural amino acid at the specified position.
1003531 Non-natural amino acids can also be encoded with rare codons. For example, when the arginine concentration in an in vitro protein synthesis reaction is reduced, the rare arginine codon, AGG, has proven to be efficient for insertion of Ala by a synthetic tRNA acylated with alanine.
See, e.g., Ma et al., Biochenustry, 32:7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNAArg, which exists as a minor species in Escherichia coli. Some organisms do not use all triplet codons. An unassigned codon AGA
in Micrococcus luteus has been utilized for insertion of amino acids in an in vitro transcription/translation extract. See, e.g., Kowal and Oliver, Nucl. Acid. Res., 25:4685 (1997). In one embodiment, components of the compositions and methods described herein can be generated to use these rare codons in vivo.
1003541 The incorporation of non-natural amino acids in vivo can be done without significant perturbation of the eukaryotic host cell. For example, because the suppression efficiency for the UAG codon depends upon the competition between the O-tRNA, including but not limited to, the amber suppressor tRNA, and a eukaryotic release factor (including but not limited to, eRF) (which binds to a stop codon and initiates release of the growing peptide from the ribosome), the suppression efficiency can be modulated by, including but not limited to, increasing the expression level of O-tRNA, and/or the suppressor tRNA.
1003551 Selector codoiis also comprise extended codons, including but not limited to, four or more base codons, such as, four, five, six or more base codons. Examples of four base codons include, but are not limited to, AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like. A feature of the methods and compositions described herein includes using extended codons based on frameshift suppression. Four or more base codons can insert, including but not limited to, one or multiple non-natural amino acids into the same protein. For example, in the presence of mutated O-tRNAs, including but.not limited to, a special frameshift suppressor tRNAs, with anticodon loops, for example, with at least 8-10 nt anticodon loops, the four or more base codon is read as single amino acid. In other embodiments, the anticodon loops can decode, including but not liniited to, at least a four-base codon, at least a five-base codon, or at least a six-base codon or more. Since there are 256 possible four-base codons, multiple non-natural amino acids can be encoded in the same cell using a four or niore base codon. See, Anderson et al., (2002) Exploring the Limits of Codon and Anticodon Size, Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:
Selection of Effrcient Suppressors of Four-base Codoais and Identifcation of "Shifty" Four-base Codons with a Library Approach in Escherichia coli, J. Mol. Biol. 307: 755-769.
(00356) For example, four-base codons have been used to incorporate non-natural amino acids into proteins using in vitro biosynthetic methods. See, e.g., Ma et al., (1993) Biochemistry, 32:7939-7945; and Hohsaka et al., (]999) J. Am. Chem. Soc., .121:34-40. CGGG and AGGU were used to simultaneously incorporate 2-naphthylalanine and an NBD derivative of lysine into, streptavidin in vitro with two chemically acylated frameshift suppressor tRNAs. See, e:g., Hohsaka et al., (I 999). J. Am. Chem.
Soc., 121:12194-12195. In an in vivo study, Moore et al. examined the ability of tRNALeu derivatives with NCUA
anticodons to suppress UAGN codons (N can be U, A, G, or C), and found that the quadruplet UAGA can be decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13 to 26% with little decoding in the 0 or -1 frame. See, Moore et al., (2000) J. Mol. Biol., 298:195-205. In one embodiment, extended codons based on rare codons or nonsense codons can be used in the methods and compositions described herein, which can reduce missense readthrough and frameshift suppression at other unwanted sites.
1003571 For a given system, a selector codon can also include one of the natural three base codons, where the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes a system that is lacking a tRNA that recognizes the natural three base codon, and/or a system where the three base codon is a rare codon.
1003581 Selector codons optionally include unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125. Properties of third base pairs include stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by a polymerase, and the efficient continued primer extension after synthesis of the nascent unnatural base pair. Descriptions of unnatural base pairs which can be adapted for methods and compositions include, e.g., Hirao, et al., (2002) An unnatural base pair for incorporating amino acid analogues into protein, Nature Biotechnology, 20:177-182, and see also, Wu, Y., et. al. (2002) J: Am.
Chem. Soc. 124:14626-14630.
Other relevant publications are listed herein.

100359] For in vivo usage, the unnatural nucleoside is membrane permeable and is phosphorylated to form the corresponding triphosphate. In addition, the increased genetic information is stable and not destroyed by cellular enzymes. Previous efforts by Benner and others took advantage. of hydrogen bonding patterns that are different from those in canonical Watson-Crick pairs, the most noteworthy example of which is the iso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc., 111:8322-8322; and Piccirilli et al., (1990) Nature, 343:33-37;
Kool, (2000) Curr. Opin. Chem. Biol., 4:602-608. These bases in general mispair to some degree with natural bases and cannot be enzytnatically replicated. Kool and co-workers demonstrated that hydrophobic packing.

interactions between bases can replace hydrogen bonding to drive the formation of base pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602-608; and Guckian and Kool, (1998) Angew. Chem.
Int. Ed. Engl.,.36(24): 2825-2828. In an effort to develop an unnatural base pair satisfying all the above requirements, Schultz, Romesberg and co-workers have systematically synthesized and studied a series of unnatural hydrophobic bases. A
PICS:PICS self-pair is found to be more stable than natural base pairs, and can be efficiently incorporated into DNA by Klenow fragment of Escherichia coli DNA polymerase I(KF). See, e.g., McMinn et al., (1999) J. Am.
Chem. Soc., 121:11585-11586; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274-3278. A 3MN:3MN self-pair can be synthesized by KF with efficiency and selectivity sufficient for biological function. See, e.g., Ogawa et al., (2000) J. Am. Chem. Soc., 122:8803-8804. However, both bases act as a chain terminator for further replication. A mutant DNA polymerase has been recently evolved that can be used to replicate the PICS self pair. In addition, a 7AI self pair can be replicated. See, e.g., Tae et al., (2001) J. Am. Chem. Soc., 123:7439-7440. A novel metallobase pair, Dipic:Py, has also been developed, which fonns a stable pair upon binding Cu(II). See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714-10715.
Because extended codons and unnatural codons are intrinsically orthogonal to natural codons, the non-natural amino acid methods described herein can take advantage of this property to generate orthogonal tRNAs for them.

1003601 A translational bypassing system can also be used to incorporate a non-natural amino acid in a desired polypeptide. In a translational bypassing system, a large sequence is incorporated into a gene but is not translated into protein. The sequence contains a structure that serves as a.cue io induce the ribosome to hop over the sequence and resume translation downstream of the insertion.
1003611 In certain embodiments, the protein or polypeptide of interest (or portion thereof) in the methods and/or compositions described herein is encoded by a nucleic acid. Typically, the nucleic acid comprises at least one selector codon, at least two selector codons, at least three selector codons, at least four selector codons, at least five selector codons, at least six selector codons, at least seven selector codons, at least eight selector codons, at least nine selector codons, ten or more selector codons.
1003621 Genes coding for proteins or polypeptides of interest can be mutagenized using, for exaniple, methods described herein under "Mutagenesis and Other Molecular Biology Techniques" to include, for example, one or more selector codons for the incorporation of a non-natural amino acid. For example, a nucleic acid for a protein of interest is mutagenized to include one or more selector codons, providing for the incorporation of the one or more non-natural amino acids. The methods and conipositions described herein include any such variant, including but not limited to, niutant, versions of any protein, for example, including at least one non-natural amino acid. Similarly, the methods and compositions described herein include corresponding nucleic acids, i.e., any nucleic acid with one or more selector codons that encodes or allows for the in vivo incorporation of one or more non-natural amino acid.
1003631 Nucleic acid molecules encoding a polypeptide of interest, including by way of example only, GH
polypeptide may be readily mutated to introduce a cysteine at any desired position -of the polypeptide. Cysteine is widely used to introduce reactive molecules, water soluble polymers, proteins, or a wide variety of other molecules, onto a protein of interest. Methods suitable for the incorporation of cysteine into a desired position of a polypeptide include those described in U.S. Patent No. 6,608,183, which is herein incorporated by reference for the aforementioned disclosure, and mutagenesis techniques. The use of such cysteine-introducing and utilizing techniques can be used in conjunction with the non-natural amino acid introducing and utilizing techniques described herein.
VIIL Ii: vivo generation of polypeptides con:prising non-natural amino acids 1003641 For convenience, the in vivo generation of polypeptides comprising non-natural amino acids described in this section have been described generically and/or with specific examples.
However, the in vivo generation of polypeptides comprising non-natural amino acids described in this section should not be limited to just the generic descriptions or specific example provided in this section, but rather the in vivo generation of polypeptides comprising.non-natural amino acids described in'this section apply equally well to.all compounds that fall within the scope of Fonnulas I-XV, including any sub-formulas or specific compounds that fall within the scope of Formulas I-XV that are described in the specification, claims and figures herein.
1003651 The polypeptides described herein can be generated in vivo using modified tRNA and tRNA
synthetases to add to or substitute amino acids that are notencoded in naturally-occurring systems.
1003661 Methods for generating tRNAs and tRNA synthetases which use amino acids that are not encoded in naturally-occurring systems are described in, e.g., U.S. Patent No. 7,045,337, entitled "In vivo incorporation of unnatural amino acids" and U.S. Patent No. 7,083,970, entitled "Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs" which are incorporated by reference herein. These niethods involve generating a translational machinery that functions independently of the synthetases and tRNAs endogenous to the translation system (and are therefore sometimes referred to as "orthogonal"). In'one embodinient the translation system comprises a polynucleotide encoding the polypeptide; the. polynucleotide can be mR.NA that was transcribed from the corresponding DNA, or the mRNA may arise from an RNA viral vector; further the polynucleotide comprises a selector codon corresponding to the predesignated site of incorporation for the non-natural amino acid. The translation further comprises a tRNA comprising the non-natural amino acid, where the tRNA is specific to the aforementioned selector codon;. in further embodiments, the non-natural amino acid is aminoacylated. In further or additional embodiments, the translation system comprises an aminoacyl synthetase specific for the tRNA; and in other or further embodiments, the translation system comprises an orthogonal tRNA and an orthogonal anzinoacyl tRNA
synthetase. In further or additiorial embodiments, the -translation system comprises at least one of the following:
a plasmid comprising the aforementioned polynucleotide (typically in the form of DNA), genomic DI`TA
comprising the aforementioned polynucleotide (typically in the form of DNA), or genomic DNA into which the-aforementioned polynucleotide has been integrated (in further embodiments, the integration is stable integration). In further or additional embodiments of the translation system, the selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon. In further or additional embodiments of the translation system, the tRNA is a suppressor tRNA. In further or additional embodiments, the non-natural amino acid polypeptide is synthesized by a ribosome.
1003671 In further or additional embodiments, the translation system comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS). Typically, the O-RS
preferentially anunoacylates the 0-tRNA with at least one non-natural amino acid in the translation system and the O-tRNA recognizes at least one selector codon that is not recognized by other tRNAs in the system. The translation system thus inserts the non-natural amino acid into a polypeptide produced in the system, in response to an encoded selector codon, thereby "substituting" a, non-natural amino acid into a position in the encoded polypeptide.

1003681 A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases for inscrting particular synthetic amino acids into polypeptides are generally suitable for use in the methods described herein to produce the non-natural amino acid polypeptides described herein. For example, keto-specific O-tRNA/aminoacyl-tRNA
synthetases are described in Wang, L., et al., Proc. Natl. Acad. Sci. USA
l00(i):56-61 (2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). Exemplary O-RS, or portions thereof, are encoded by polynucleotide sequences and include amino acid sequences disclosed in U.S. Patent Nos.
7,045,337 and 7,083,970, each incorporated herein by reference in their entirety. Corresponding O-tRNA
molecules for use with the O-RSs are also described in U.S. Patent Nos. 7,045,337 and 7,083,970 which are incorporated by reference in their entirety herein. In addition, Mehl et al. in J. Am. Chem Soc. 2003; 125:935-939 and Santoro et al. Nature Biotechnology 2002 Oct; 20:1044-1048, which are incorporated by reference in their entirety herein, discuss screening methods and aminoacyl tRNA synthetase and tRNA molecules for the incorporation of p-aniinophenylalanine into polypeptides 1003691 Exemplary O-tRNA sequences suitable for use in the methods described herein include, but are not limited to, nucleotide sequences SEQ ID NOs: I or 2 as disclosed in U.S.
Patent No. 7,045,337 which is incorporated by reference herein. Other examples of O-tRNA/aminoacyl-tRNA
synthetase pairs specific to particular non-natural amino acids are described in U.S. Patent No. 7,083,970 which is incorporated by reference in its entirety herein. O-RS and O-tRNA that incorporate both keto-and azide-containing amino acids in S. cerevisiae are described in Chin, J. W., et al., Science 301:964-967 (2003).
1003701 Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of a specific codon which encodes the non-natural amino acid. While any codon can be used, it is generally desirable to select a codon that is rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNA synthetase is expressed. By way of example only, exeniplary codons include nonsense codon such as stop codons (amber, ochre, and opal), four or more base codons and other natural three-base codons that are rarely or unused.
1003711 Specific selector codon(s) can be introduced into appropriate positions in the polynucleotide coding sequence using mutagenesis methods (including but not limited to, site-specific mutagenesis, cassette mutagenesis, restriction selection mutagenesis, etc.).
1003721 Methods for generating components of the protein biosynthetic machinery, such as O-RSs, O-tRNAs, and ortliogonal O-tRNA/O-RS pairs that can be used to incorporate a non-natural amino acid are described in Wang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J. Am.
Chem Soc. 124:9026-9027 (2002);
Zhang, Z. et al., Biochemistry 42: 6735-6746 (2003). Methods and compositions for the in vivo incorporation of non-natural amino acids are dcscribed in U.S. Patent No. 7,045,337 which is incorporated by reference in its entirety herein. Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use in vivo translation system of an organism are also described in U.S. Patent Nos. 7,045,337 and 7,083,970 which are incorporated by reference in its entirety herein. In addition PCT Publication No. WO
04/035743 entitled "Site Specific Incorporation of Keto Amino Acids into proteins, which is incorporated by reference in its entirety, describes orthogonal RS and tRNA pairs for the incorporation of keto amino acids. PCT
Publication No. WO 04/094593 entitled "Expanding the Eukaryotic Genetic Code," which is incorporated by reference herein in its entirety, describes orthogonal RS and tRNA pairs for the incorporation of non-naturally encoded amino acids in eukaryotic host cells.

1003731 Methods for producing at -least. one recombinant orthogonal aminoacyl-tRNA syitthetase (O-RS) contprise; (a) generating a library .of (optionally mutant) RSs derived from at least one aminoacyl-tRNA
synthetase (RS) from a first organism, including but not liniited to, a prokaryotic organism, such as, by way of example only, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P. firriosus, P. horikoshii, A. pernix. T.
thermaphilus, or the like, or a eukaryotic organism; (b) selecting (and/or screening), the library of RSs (optionally mutant RSs) for meinbers that anunoacylate an orthogonal tRNA (O-tRNA) in the presence of a non-natural atnino acid and a natural amino acid, thereby providing a pool of active (optionally mutant) RSs; and/or,_ (c) selecting (optionally through negative selection) the pool for active RSs (including but not limited to, mutant RSs) that preferentially aminoacylate the O-tRNA in the absence of the non-natural anuno acid, thereby providing the at least one recombinant O-RS; wherein the at least one recombinant O-RS preferentially aminoacylates the O-tRNA witli the non-natural amino acid.
1003741 In one embodiment, the RS is an inactive RS. The inactive RS can be generated by mutating an active RS. By way of example only, the inactive RS can be generated by mutating at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, or at least about 10 or more amino acids to different anuno acids, including but not limited,to, alanine.
[003751 Libraries of mutant RSs can be generated using various techniques, including but not.limited to rational design based on protein three dimensional RS structure, or mutagenesis of RS
nucleotides in a random or rational design technique. By way of example only, the mutant RSs can be generated by site-specific mutations, random mutations, diversity generating recombination mutations, chimeric constructs, rational design and by other methods described herein.
(003761 In one embodiment, selecting (and/or screening) the library of RSs (optionally mutant RSs) for members that are active, including but not limited to, those which aniinoacylate an orthogonal tRNA (O-tRNA) in the presence of a non-natural amino acid.and a natural amino acid, includes, but is not limited to: introducing a positive selection or screening marker, including but not limited to, an antibiotic resistance gene, or the like, and the library of (optionally mutant) RSs into a plurality of cells, wherein the positive selection and/or screening marker comprises at least one selector codori, including but not limited to, an amber codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon;
growing the plurality of cells in the presence of a selection agent;
identifying cells that survive (or show a specific response) in the presence of the selection andlor screening agent:by suppressing the at least one selector codon in the positive selection or screening marker, thereby providing a subset of positively selected cells that contains the pool of active (optionally mutant) RSs. Optionally, the selection and/or screening agent concentration can be varied.
1003771 In one aspect, the positive selection marker is_a chloramphenicol acetyltransferase (CAT) gene and the selector codon is an amber stop codon in the CAT gene. Additional selection markers include, but are not limited to, a neomycin resistance gene, a blasticidin resistance gene, a hygromycin resistance gene, or any other available resistance genes. Optionally, the positive selection marker is a(3-lactamase gene and the selector codon is an atnber stop codon in the (3-lactamase gene. In another aspect the positive screening marker comprises a fluorescent or luminescent screening marker or an affinity based screening marker (including but not limited to, a cell surface marker).

1003781 In one embodiment, negatively selecting or screening the pool for active RSs (optionally mutants) including, but not limited to, those which preferentially aminoacylate the O-tRNA in the absence of the non-natural amino acid includes, but is not limited to: introducing a negative selectiotror screening marker with the pool of active (optionally mutant) RSs from the positive selection or screening into a plurality of cells of a second organism, wherein the negative selection or screening marker comprises at least one selector codon (including but not limited to, an -antibiotic resistance gene, including but not liniited to, a chloramphenicol acetyltransferase (CAT) gene); and, identifying cells that survive or show a specific screening response in a first medium supplemented with the non-natural amino acid and a screening or selection agent, but fail to survive or to show the specific response in a second medium not supplemented with the non-natural amino acid and the selection or screening agent, thereby providing surviving cells or screened cells with the at least one recombinant O-RS. By way of example only, a CAT identification protocol optionally acts as a positive selection and/or a negative screening in determination of appropriate O-RS
recombinants. For instance, a pool of clones is optionally replicated on growth plates containing CAT (which comprises at least one selector codon) either with or without one or more non-natural amino acid. Colonies growing exclusively on the plates containing non-natural amino acids are thus regarded as containing recombinant O-RS. In one aspect, the concentration of the selection (and/or screening) agent is varied. In some aspects the first and second organisms are different. Thus, the first and/or second organism optionally comprises: a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungi, a yeast, an archaebacterium, a eubacterium, a plant, an insect, a protist, etc. In other embodiments, the screening marker comprises a fluorescent or lunvnescent screening marker or an affinity based screening niarker.
1003791 In another embodiment, screening or selecting (including but not limited to, negati,vely selecting) the pool for active (optionally mutant) RSs includes, but is not limited to:
isolating the pool of active mutant RSs from the positive selection step (b); introducing a negative selection or screening marker, wherein the negative selection or screening marker comprises at least one selector codon (including but not limited to, a toxic marker gene, including but not limited to, a ribonuclease barnase gene, comprising at least one selector codon), and the pool of active (optionally mutant) RSs into a plurality of cells of a second organism; and identifying cells that survive or show a specific screening response in a first medium not supplemented witlt the non-natural amino acid, but fail to survive or show a specific screening response in a second medium supplemented with the non-natural amino acid, thereby providing surviving or screened cells with the at least one recombinant O-RS, wlierein the at least one recombinant O-RS is specific for the non-natural amino acid. In one aspect, the at least one selector codon comprises about two or more selector codons. Such embodiments optionally can include wherein the at least one selector codon comprises two or more selector codons, and wherein the first and second organism are different (including but not limited to, each organism is optionally, including but not limited to, a prokaryote, a eukaryote, a mammal, an Escher=ichiu. coli, a fungi, a yeast, an archaebacteria, a eubacteria, a plant, an irtsect, a protist, etc.). Also, some aspects include wherein the negative selection marker comprises a ribonuclease barnase gene (which comprises at least one selector codon). Other aspects include wherein the screening niarker optionally comprises a fluorescent or luminescent screening marker or an affinity based screening marker. In the embodiments herein, the screenings and/or selections optionally include variation of the screening and/or selection stringency.

1003801 In another embodiment, the methods for producing at least one recombinant orthogonal aminoacyl-tRNA synthetase (O-RS) may further comprise: (d) isolating the at least one reeombinant O-RS; (e) generating a second set of O-RS (optionally mutated) derived from the at least one recombinant O-RS; and, (f) repeating steps (b) and (c) until a inutated O-RS is obtained that comprises an ability to preferentially aminoacylate the 0-tRNA. Optionally, steps (d)-(f) are repeated, including but not limited to, at least about two times. In one aspect, the second-set of mutated O-RS derived from at,least one.recombinant O-RS.oan be generated by mutagenesis, including but not limited to, random mutagenesis, site-specific mutagenesis,.
recombination or a combination thereof 1003811 The stringency of the selection/screening steps, including but not limited to, the positive selection/screening step (b), the negative selection/screening step (c) or both the positive and negative selection/screening steps (b) and (c), in the above-described methods, optionally includes varying the selectionlscreening stringency. In another embodiment, the positive selection/screening step (b), the negative selection/screening step (c) or both the positive and negative selection/screening steps (b) and (c) comprise using a reporter, wherein the reporter is detected by fluorescence-activated cell sorting (FACS) or wherein the reporter is detected by luminescence. Optionally, the reporter is displayed on a cell surface, on a phage display or the like and selected based upon affinity or catalytic activity involving the non-natural amino acid or an analogue. In one embodinient, the mutated synthetase is displayed on a cell surface, on a phage display or the like.
1003821 Methods for producing a recombinant orthogonal tRNA (O-tRNA) include, but are not limited to: (a) generating a library ofmutant tRNAs derived from:at least one tRNA, including but not limited to, a suppressor tRNA, from a first organism; (b) selecting (including but not limited to, negatively selecting) or screening the library for (optionally mutant) tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) froni a second organisin in the absence'of a RS from the first organisni, thereby providing a pool of tRNAs (optionally mutant); and, (c) selecting or screening the pool of tRNAs (optionally mutant) for inenibers that are aminoacylated by an introduced orthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA;
wherein the at least one recombinant .0-tRNA recognizes-a selector codon and is not efficiency recognized by the RS from the.second organism and is preferentially aminoacylated by the O-RS. In some embodiments the at least one tRNA is a suppressor tRNA and/or comprises a. wiique three base codon of natural and/or unnatural bases, or is a nonsense codon, a rare codon, an unnatural codon, :a codon comprising at least 4 bases, an aniber codon, an ochre codon, or an opal stop codon. In one embodiment, the recoinbinant O-tRNA possesses an improvement of orthogonality. It will be appreciated that in some embodiments, O-tRNA is optionally imported into a first organism from a second organism without the need for modification. In various embodiments, the first and second organisms are either the same or different and are optionally chosen from, including but not limited to, prokaryotes (including but not limited to, Methanococcus jannaschii, Metlranobacteriuni thern7oautotrophicuni, Escherichia coli, Halobacteriuni, etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria, plants, insects, protists, etc. Additionally, the recombinant tRNA is optionally aniinoacylated by a non-natural amino acid, wherein the non-natural amino acid is biosynthesized in vivo either naturally or through genetic manipulation. The non-natural amino acid is optionally added to a growth medium for.at least the first or second organism, wherein the non-natural amino acid is capable of achieving appropriate intracellular concentrations to allow incorporation into the non-natural anuno acid polypeptide 1003831 In one aspect, selecting (including but not liniited to, negatively selecting) or screening the library for (optionally mutant) tRNAs that are aminoacylated by an aminoacyl-tRNA
synthetase (step (b)) includes:
introducing a toxic marker gene, wherein the toxic marker gene comprises at least one of the selector codons (or a gene that leads to the production of a toxic or static agent or a gene essential to the organism wherein such marker gene comprises at least one selector codon) and the library of (optionally mutant) tRNAs into a plurality of cells from the second organism; and, selecting surviving cells, wherein the surviving cells contain the pool of (optionally mutant) tRNAs comprising at least one orthogonal tRNA or nonfunctional tRNA. For example, surviving cells can be selected by using a comparison ratio cell density assay.
1003841 In another aspect, the toxic marker gene can include two or more selector codons. In another embodiment of the. methods described herein, the toxic marker gene is a ribonuclease baniase gene, where the ribonuclease bamase gene comprises at least one amber codon. Optionally, theribonuclease barnase gene can include two or more amber codons.
1003851 In another embodiment, selecting or screening the pool of (optionally mutant) tRNAs for members that are aminoacylated by an introduced orthogonal RS (O-RS) can include:
introducing a positive selection or screening marker gene, wherein the positive marker.gene. comprises a drug resistance gene (including but not liniited to, 0-lactamase gene, comprising at least one of ihe selector codons, such as at least one amber stop codon) or a gene essential to the organism, or a gene that leads to detoxification of a toxic agent, along with the O-RS, and the pool of (optionally mutant) tRNAs into a plurality of cells from the second organism; and, identifying surviving or screened cells grown in the presence of a selection or screening agent, including but not lirr-ited to, an antibiotic, thereby providing a pool of cells possessing the at least one recombinant tRNA, where the at least one recombinant tRNA is aminoacylated by the O-RS and inserts an amino acid into a translation product encoded by the positive marker gene, in response to the at least one selector codons. In another enibodiment, the concentration of the selection and/or screening agent is varied.
1003861 Methods for generating specific O-tRNA/O-RS pairs are provided.
Methods include, but are not limited to: (a) generating a library of mutant tRNAs derived from at least one tRNA from a first organism; (b) negatively selecting or screening the library for (optionally mutant) tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of =a RS
from the first organism, thereby providing a pool of (optionally mutant) tRNAs; (c) selecting or screening the pool of (optionally mutant) tRNAs for members that are aminoacylated by an introduced orthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA. The at least one recombinant O-tRNA
recognizes a selector codon and is not efficiently recognized by the RS from the second organism and is prefereintially aminoacylated by the O-RS.
The method also includes (d) generating a library of (optionally mutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS) from a third organism; (e) selecting or screening the library of mutant RSs for members that preferentially aminoacylate the at least one recombinant O-tRNA
in the presence of a non-natural anvno acid and a natural aniino acid, thereby providing a pool of active (optionally mutant) RSs; and, (f) negatively selecting or screening the pool for active (optionally mutant) RSs that preferentially aminoacylate the at least one reconibinant O-tRNA in the absence of the non-natural amino acid, thereby providing the at least one specific O-tRNA/O-RS pair, wherein the at least one specific O-tRNA/O-RS
pair comprises at least one reconibinant O-RS that is specific for the non-natural anuno acid and the at least one recombinant O-tRNA.
Specific O-tRNA/O-RS pairs produced by the methods described herein are included within the scope and methods described herein. For example, the specific O-tRNA/O-RS pair can include, including but not limited to, a mutRNATyr-mutTyrRS pair, such as a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, a mutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.
Additionally,, such methods include wherein the first and third organism are the same (including but not limited to, Methanococcus jannaschit).
1003871 Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use in an in vivo translation system of a second organism are also included in the methods described herein.
The methods include, but are not limited to: introducing a. marker gene, a tRNA and an aminoacyl-tRNA
synthetase (RS) isolated or derived from a first organism into a first set of cells from the second organism;
introducing the marker gene and the tRNA into a duplicate cell set from.a second organism; and, selecting for surviving cells in the first set that fail to survive in the duplicate cell set or screening for cells shoxving a specific screening response that fail to give such response in the duplicate cell set, wherein the first set and the duplicate cell set are grown in the presence of a selection or screening agent, wherein the surviving or screened cells comprise the orthogonal tRNA-tRNA
synthetase pair for use in the in.the in vivo translation system of -he second organism. In one embodiment, comparing and selecting or screening includes an in vivo coniplementation assay. The concentration of the selection or screening agent can be varied.
(00388] The organisms described herein comprise a variety of organism and a, variety of combinations. In one embodiment, the organisms are optionally a prokaryotic organism, including but not limited to, Methanococcus jannaschii, /l9ethanobacteriunt thermoautotrophicunt; Halobacterium, Escherichia coli, A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophiltis, or -the.like. Alternatively, the organisms are a eukaryotic organism, including but not limited to, plants (including but not linmited to, complex plants such as monocots, or dicots), algae, protists, fungi (including but not limited to, yeast, etc), animals (including but not limited to, manunals, insects, arthropods, etc.), or the like.

A. Expression in Non-eukaryotes and Eukaryotes 1003891 The techniques disclosed in this section can be applied to the expression in non-eukaryotes and eukaryotes of the non-natural amino acid polypeptides described herein. Such expression systems are further described in U.S. Patent Application No.
1003901 A eukaryotic host cell or non-eukaryotic host cell as described herein provides the ability to synthesize polypeptides which comprise non-natural amino acids in large useful quantities. In one aspect, the composition optionally includes, but is not litnited to, at least 10 tnicrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least one gram, or more of the polypeptide that comprises a non-natural amino acid, or an amount that can be achieved with in vivo polypeptide production methods (details on recombinant protein production and purification are provided herein).. In another aspect, the polypeptide is optionally present- in the composition at a concentration of, including but not limited to, at least 10 niicrograms of polypeptide per liter, at least 50 micrograms of polypeptide per liter, at least 75 nucrograms of polypeptide per liter, at least 100 micrograms of polypeptide per liter, at least 200 microgrants of polypeptide per liter, at 7east 250 nucrograms of polypeptide per liter, at least 500 mictograms of polypeptide per liter, at least 1 milligram of polypeptide per liter, or at least 10 milligrams of polypeptide per liter or more, in, including but not limited to, a cell lysate, a buffer, a pharmaceutical buffer, or other liquid suspension (including but not liniited to, in a volume of anywhere from about 1 nl to about 100 L or more).
The production of large quantities (including but not limited to, greater that than typical. with other methods, including but not limited to, in vitro translation) of a protein in a eukaryotic cell including at least one non-natural aniino acid is a feature of the methods, techniques and compositions described herein.
1003911 A eukaryotic host cell or non-eukaryotic host cell as described herein provides the ability to biosynthesize polypeptides that comprise non-natural amino acids in large useful quantities. For example, polypeptides comprising a non-natural aniino acid can be produced at a concentration of, including but not limited to, at least 10 g/liter, at least 50 pg/liter, at least 75 pg/liter, at.least 100 g/liter, at least 200 pg/liter, at least 250 pg/liter, or at least 500 g/liter, at least lmg/liter, at least 2mg/liter, at least 3 mg/liter, at least 4 mg/liter, at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least 8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400. 500, 600, 700, 800, 900 mg/liter, I g/liter, 5 g/liter, 10 g/liter or more of protein in a cell extract, cell lysate, culture medium, a buffer, and/or the like.
[003921 The techniques disclosed in this section can be applied to the expression systems, culture and isolation of the non-natural amino acid polypeptides described herein. Non-natural amino acid polypeptides may be expressed in any number of suitable expression systenis including, but not limited to, yeast, insect cells, mammalian cells, and bacteria.
1003931 Once a recombinant host cell strain has been established (i.e., the expression construct has been introduced into the host cell and host cells with the proper expression constntct are isolated), the recombinant host cell strain is cultured under conditions appropriate for production of polypeptides. The method of culture of the recombinant host cell strain will be dependent on the nature of the expression construct utilized and the identity of the host cell. Recombinant host strains are normally cultured using documented methodologies.
Recombinant ltost cells are typically cultured in liquid medium containing assimilatable sources of carbon, nitrogen, and inorganic salts and, optionally, containing vitaniins, amino acids, growth factors, and other proteinaceous culture supplements. Liquid media for culture of host cells may optionally contain antibiotics or anti-fungals to prevent the growth of undesirable microorganisms and/or compounds including, but not limited to, antibiotics to select for host cells containing the expression vector.
1003941 Recombinant host cells may be cultured in batch or continuous formats, with either cell harvesting (in the case where the desired polypeptide accumulates intracellularly) or harvesting of culture supematant in either batch or continuous formats. For production in prokaryotic host cells, batch culture and cell harvest are preferred. Where protein expression is accomplished via a cell or cell line expression system, cells can be propagated in vitro in a variety of modes including, but not limited to, non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth). Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products. Again, cell type and propagation mode may be selected based on a variety of production considerations as described above.
1003951 In one embodinient, the non-natural amino acid polypeptides described herein are purified after expression in recombinant systenis. The polypcptides may be ptirified from host cells or culture medium by a variety of documented methodologies. Normally, many polypeptides produced in bacterial host cells are poorly soluble or insoluble (in the form of inclusion bodies). In one embodiment, anuno acid substitutions are readily made in the polypeptides that are selected for the purpose of increasing the solubility of the recombinantly produced polypeptide utilizing the methods disclosed herein. In the case of insoluble polypeptides, the polypeptides may be collected from host cell lysates by centrifugation or filtering and may further be followed by homogenization of the cells. In the case of poorly soluble polypeptides, compounds including, but not limited to, polyethylene imine (PEI) may be added to induce . the precipitation of partially soluble polypeptides.
The precipitated polypeptides may then be conveniently collected by centrifugation or filtering. Recombinant host cells may be disrupted or homogenized to release the inclusion bodies from within the cells using documented methods. Host cell disruption or homogenization may be performed using well documented methodologies including, but not liniited to, enzymatic cell disruption, sonication, dounce homogenization, or high pressure release disruption. In one embodiment of the methods described and encompassed herein, the high pressure release technique is used to disrupt the E. coli host cells to release the inclusion bodies of the polypeptides. When hatidling inclusion bodies of polypeptides, it is advantageous to minitnize the Itomogenization time on repetitions in order to maximize the yield of inclusion bodies without loss due to factors such as solubilization, mechanical shearing or proteolysis.
(00396) Insoluble or precipitated polypeptides may then be solubilized using any of a number of documented suitable solubilization agents. By way of example, the polypeptides are solubilized with urea or guanidine hydrochloride. The volume of the solubilized polypeptides should be.
minimized. so that large batches may be produced using'conveniently manageablebatch sizes. This factor may be significant in a large-scale cotnmercial setting where the recombinant host may be grown in batches that are thousands of liters in volume. In addition, when manufacturing polypeptides in a large-scale commercial setting, in particular for human pharmaceutical uses, the avoidance of harsh chemicals that can damage the machinery and container, or the polypeptide product itself, should be avoided, if possible. It has been shown in the methods described and encompassed herein that the milder denaturing agent urea can be used to solubilize the polypeptide inclusion bodies in place of the harsher denaturing agent guanidine hydrochloride. The use of urea significantly reduces the risk of damage to stainless steel equipment utilized in the manufacturing and purification process of a polypeptide while efficiently solubilizing the polypeptide inclusion bodies.
1003971 In the case of soluble polypeptides, the peptides may be secreted into the periplasmic space or into the culture mediurn. In addition, soluble, peptides may be present. in the cytoplasm of the host cells. The soluble peptide may be concentrated prior to perfonning, purification steps.
documented niethodologies, including but not limited to those described herein, may be used to concentrate soluble peptide from, by way of example, cell lysates or culture medium. In addition, documented methodologies,. including, but not limited to those described lterein, may be used to disrupt host cells and release soluble peptide from the cytoplasm or periplasmic space of the host cells.
1003981 When the polypeptide is produced as a fusion protein, the, fusion sequerice is preferably removed.
Removal of a fusion sequence may be accomplished by tnethods including, but not limited to, enzymatic or chemical cleavage, wherein etizymatic. cleavage is preferred. Enzymatic removal of fusion sequences may be accomplished using-documented.methodologies. The choice of enzyme for removal of the fusion sequence will be determined by the identity of the fusion, and the reaction conditions will be specified by the choice of enzyme. Chemical cleavage may be accomplished using reagents, including but not limited to, cyanogen broniide, TEV protease, and other reagents. The cleaved polypeptide is optionally purified froni the cleaved fusion sequence by documented methodologies. Such methods will be determined by the identity and properties of the fusion sequence and the polypeptide. Methods for purification may include, but are not limited to, size-exclusion chromatography, hydrophobic interaction chrotnatography, ion-exchange chroniatography or dialysis or any combination thereof.

1003991 The polypeptide is also optionally purified to remove DNA from the protein solution. DNA may be removed by documented methodologies, including, but not liniited to, precipitation or ion exchange chromatography. In one embodiment, DNA is removed by precipitation with a nucleic acid precipitating agent, such as, but not limited to, protamine sulfate. The polypeptide may be separated from die precipitated DNA
using documented niethodologies including, but not limited to, centrifugation or filtration. Removal of host nucleic acid molecules is an-important factor in a setting where the polypeptide is to be used to treat humans and the methods described herein reduce host cell DNA to pharmaceutically acceptable levels.
1004001 Methods for small-scale or large-scale fermentation may also be used in protein expression, including but not limited to, fermentors, shake flasks, fluidized bed bioreactors, hollow fiber bioreactors, roller bottle culture systems, and stirred tank bioreactor systems. Each of these methods can be performed in a batch, fed-batch, or continuous niode process.
1004011 Human forms of the non-natural amino acid polypeptides described herein can generally be recovered using documented methodologies. For example, culture medium or cell lysate can be centrifuged or filtered to reinove cellular debris. The supernatant may be concentrated or diluted to a desired volume or diafiltered into a suitable buffer to condition the preparation for further purification. Further purification of the non-natural anuno acid polypeptides described herein include, but are not limited to, separating deamidated and clipped forms of a potypeptide variant from the corresponding intact form.
1004021 Polypeptides encompassed within the methods and compositions described herein, including but not limited to, polypeptides comprising non-natural amino acids, antibodies to polypeptides comprising non-natural amino acids, binding partners for polypeptides comprising non-natural aniino acids, may be purified, either partially or substantially to homogeneity, according to documented inethodologies. Accordingly, polypeptides described herein may be recovered and purified by documented methodologies, including but not limited to, amrnonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography, gel electrophoresis and any combination thereof. Protein refolding steps can be used, as desired, in making correctly folded mature proteins. High performance liqiiid chromatography (HPLC), affinity chromatography or other suitable methods can be eniployed in final purification steps where high purity is desired. In one embodiment, antibodies made against non-natural amino acids (or polypeptides comprising non-natural amino acids) are used as purification reagents, including but not limited to, for affinity-based purification of polypeptides comprising one or more non-natural amino acid(s). Once purified, partially or to homogeneity, as desired, the polypeptides are optionally used for a wide variety of utilities, including but not liniited to, as assay components, therapeutics, prophylaxis, diagnostics, research reagents, and/or as immunogens for antibody production.
1004031 One advantage of producing polypeptides comprising at least one non-natural amino acid in a eukaryotic host cell or non-eukaryotic host cell is that typically the polypeptides will be folded in their native conformations. However, in certain embodiments of the methods and compositions described herein, after synthesis, expression and/or purification, the polypeptides may possess a conformation different from the desired conformations of the relevant polypeptides. In one aspect of the methods and compositions described herein, the expressed protein is optionally denattired and then renatured.
This optional denaturation and renaturation is accomplished utilizing documented methodologies, including but not liniited to, by adding a chaperonin to the polypeptide of interest, and by solubilizing the polypeptides in a chaotropic agent including, but not limited to, guanidine HCI, and utilizing protein disulfide isomerase.
1004041 In general, it is occasionally desirable to denature and reduce expressed polypeptides and then to cause the polypeptides to re-fold into the preferred conformation. By way of example, such re-folding. may be accomplished with the addition guanidine, urea, DTT, DTE, and/or a chaperonin to a translation product of interest: Methods of reducing, denaturing and renaturing proteins include, the references above, and Debinski, et al. (1993) J. Biol. Chem., 268: 14065-14070;.Kreitman and Pastan (1993) Bioconjug. Chem.,4: 581-585;:and Buchner, et al., (1992) Anal. Biochem., 205: 263=270. Debinski, et al., for example, describe the denaturation and ieduction of inclusion body proteins in guanidine-DTE. The proteins can be refolded in a redox buffer containing, including but not limited to, oxidized glutathione and L-arginine.
Refolding reagents can be flowed 'or otherwise moved into contact with the one or more polypeptide or other expression product, or vice-versa.
1004051 In the case of prokaryotic production of a non-natural amino acid polypeptide, the polypeptide thus produced tnay be misfolded and thus lacks or has reduced biological activity.
The bioactivity of the protein may be restored by "refolding". In one embodiment, a misfolded polypeptide is refolded by solubilizing (where the polypeptide is also insoluble), unfolding and reducing the polypeptide chain using, by way of example, one or more chaotropic agents (including , but not limited to, urea and/or guanidine) and a reducing agent capable of reducing disulfide bonds (including , but not linuted to, dithiothreitol, DTT
or 2-mercaptoethanol, 2-ME). At a moderate concentration of chaotrope, an oxidizing agent is then added (including, but not limited to, oxygen, cystine or cystamine), which allows the reformation of disulfide bonds. An unfolded or misfolded polypeptide may be refolded using doctunented methodologies, such as those described in U.S. Pat. Nos. 4,511,502, 4,511,503, and 4,512,922, each of which is herein incorporated by reference for the aforementioned disclosure.
The polypeptide may also be cofolded with other proteins to form beterodimers or heteromultimers. After refolding or cofolding, the polypeptide is optionally further purified.
[004061 After purification, the non-natural amino acid polypeptides may be exchanged into different buffers and/or concentrated by=documented methodologies, including, but not limited to, diafiltration and dialysis. hGH
that is provided as a single purified protein may be subject to aggregation and precipitation. In certain embodiments the purified non-natural amino acid polypeptides may be at least 90% pure (as measured by reverse phase high performance liquid chromatography, RP-HPLC, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE). In certain other embodiments the purified non-natural amino acid polypeptides may be at least 95% pure, or at least 98% pure, or at least 99%
or greater purity. Regardless of the exact numerical value, of the purity of the non-natural amino acid polypeptides, the non-natural aniino acid polypeptides is sufficiently pure for use as a pharmaceutical product or for further processing, including but not linvted to, conjugation with a water soluble polymer such as PEG.
1004071 In certain embodiments the non-natural amino acid polypeptides molecules may be used as therapeutic agents in the absence of other active ingredients or proteins (other than excipients, carriers, and stabilizers, serum albuinin and the like), and in certain embodiments the non-natural amino acid polypeptides molecules they may be complexed with another polypeptide or a polymer.
1004081 A wide variety of methods and procedures can be used to assess the yield and purity of a polypeptide containing one or more non-natural amino acids, including but not limited to, SDS-PAGE coupled with protein staining methods, inununoblotting, mass spectrometry, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), liquid chromatography/mass spectrometry, isoelectric focusing, analytical anion exchange, chromatofocusing, and circular dichroism. By way of exantple such methods and procedures for characterizing proteins include, but are not limited to, the Bradford assay, SDS-PAGE, and silver stained SDS-PAGE, coomassie stained SDS-PAGE. Additional methods include, but are not limited to, steps to remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs) which are located on the outer membrane of Gram-negative host cells, such as, for example, Escherichia coli. Methods for reducing endotoxin levels include, but are. not limited to, purification techniques using silic.a supports, glass powder or hydroxyapatite, reverse-phase, affinity, size-exclusiou, anion-exchange chromatography, hydrophobic interaction chromatography, a conibination of these methods, and the like. Modifications or additional methods may be required to remove contaminants such as co-migrating proteins from the polypeptide of interest.
Methods for measuring endotoxin levels include, but are.not limited to, Limulus Amebocyte Lysate (LAL) assays.
1004091 In certain embodiments amino acids of Formulas I-XV, including any sub-formulas or specific compounds that fall within the scope of Formulas I-XV, may be biosynthetically incorporated into polypeptides, thereby making non-natural amino acid polypeptides. In other embodiments, such amino acids are incorporated at a specific site within the polypeptide. In other embodiments, such amino acids incorporated into the polypeptide using atranslation system. In other embodiments, such translation systems comprise: (i) a polynucleotide encoding the polypeptide, wherein the polynucleotide comprises a selector codon corresponding to the pre-designated site of incorporation of the above amino acids, and (ii) a tRNA comprising the aniino acid, wherein the tRNA is specific to the selector codon. In other embodiments of such translation systems, the polynucleotide is mRNA produced in the translation system. In other embodinients of such translation systems, the translation system comprises a plasmid or a phage comprising the polynucleotide. In other embodiments of such translation systems, the translation system comprises genomic DNA
comprising the polynucleotide. In other embodiments of such-translation systems, the polynucleotide is stably integrated into the genomic DNA.
In other embodiments of such translation systems, the translation system comprises tRNA specific for a selector codon selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon. In other embodinients of such translation systems, the tRNA is a suppressor tRI`'A. In other embodiments of such translation systems, the translation system comprises a tRNA that is aminoacylated to the amino acids above. In other embodiments of such translation systems, the translation system comprises an aminoacyl synthetase specific for the tRNA. In other embodiments of such translation systems, the translation system comprises an orthogonal tRNA and an ortltogonal aminoacyl tRNA synthetase. In other embodiments of such translation systems, the polypeptide is synthesized by a ribosome, and in further embodiments the translation system is an in vivo translation system comprising a cell selected from the group consisting of a bacterial cell, archeaebacterial cell, and eukaryotic cell. In other embodiments the cell is an Escherichia coli cell, yeast cell, a cell from a species of Pseudomonas, mammalian cell, plant cell, or an insect cell. In other embodiments of such translation systems, the translation system is an in vitro translation systeni comprising cellular extract from a bacterial cell, archeaebacterial cell, or eukaryotic cell. In other embodiments, the cellular extract is from an Escherichia coli cell, a cell from a species of Pseudomonas, yeast-cell; mammalian cell, plant cell, or an insect cell. In other enibodinients at least:a portion of the polypeptide is synthesized by solid phase or solution phase peptide synthesis, or a combination thereof, while in other embodiments further comprise ligating the polypeptide to another polypeptide. In other embodiments amino acids of Formulas I-XV, including any sub-formulas or specific compounds that fall within the scope of Formulas I-XV, may be biosynthetically incorporated into polypeptides, wherein the polypeptide is a protein homologous to a therapeutic-protein.

B. In vivo Post-Translationa/ Modrfications 1004101 By producing polypeptides of interest with at least one non-natural amino acid in eukaryotic cells, such polypeptides may include eukary.otic post-translational modifications. In certain embodiments,, a polypeptide includes at least one non-natural amino acid and'at least one post-translational modification that is made in vivo by a eukaryotic cell, where the post-translational niodification is not made by a prokaryotic cell. By way of example, the post-traiislation modification includes, including but not limited to, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, glycosylation, and the like. In one aspect, the post-translational nlodification includes attachment of an oligosaccharide (including but not linuted to, (GIcNAc-Man)2-Man-GIcNAc-G1cNAc)) to an asparagine by a GlcNAc-asparagine linkage. See Table 1 which lists some examples of N-linked oligosaccharides of eukaryotic proteins (additional residues can also be present, which are not shown). In another aspect, the post-translational modification includes attachment of an oligosaccharide (including but not limited to, Gal-GaINAc, Gal-G1cNAc, etc.) to a serine or threonine by a GaINAc-serine or GaINAc-threonine linkage, or a G1cNAc-serine or a GIcNAc-threonine linkage.
SECTION 1.01 TABLE 1: EXAMPLES OF OLIGOSACCHARIDES THROUGH GIcNAc-LINKAGE
Type Base Structure Mana1-6 ARTICLE lI. / Mana1-6 High-mannose Mana1-3 > ManR1-4GIcNAc~31-4GIcNAcR1-Asn Mana1-3 Mana1-6 Article III. ~ ManR1-4GIcNAc~31-4GIcNAcR1-Asn Hybrid GIcNAcR1-2 Mana1-3 Article lv. GIcNAc(31-2 Mana1-6 Complex > Man(31-4GIcNAc(31-4GIcNAc~31-Asn GIcNAc~31-2 Mana1-3 Article V. Mana1-6 Xylose > Manp1-4GIcNAcp1-4GIcNAcR1-Asn xyl (00411 1 In yet another aspect, the post-translation modification includes proteolytic processing of precursors (including but not limited to, calcitonin precursor, calcitonin gene-related peptide precursor,. preproparathyroid hornnone, preproinsulin, proinsulin, prepro-opionielanocortin, pro-opiomelanocortin and the like), assembly into a multisubunit protein or macromolecular assembly, translation to another site in the cell (including but not limited to, to organelles, such as the endopiasmic reticulum, the golgi apparatus, the nucleus, lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., or through the secretory pathway). In certain embodiments, the protein comprises a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, or the like.
1004121 One advantage of a non-natural amino acid is that it presents additional chemical moieties that can be used to add additional molecules. These modifications can be made in vivo in a eukaryotic or non-eukaryotic cell, or in vitro. Thus, in certain embodiments, the post-translational niodification is through the non-natural amino acid. For example, the post-translational modification can be through a nucleophilic-electrophilic reaction. Most reactions currently used for the selective modification of proteins involve covalent bond formation betweeu nucleophilic and electrophilic reaction partners, including but not limited to the reaction of a-haloketones with histidine or cysteine side chains. Selectivity in these cases is determined by the number and accessibility of the nucleophilic residues in the protein. In polypeptides described herein or produced using the methods described herein, other more selective reactions can be used, including but not limited to, the reaction of a non-natural carbonyl amino acid with hydrazine, in vitro and in vivo.
Illustrative exaniples may be found in the following references. Comish, et al., (1996) J. Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science, 276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al., (2002) J. Am. Chem. Soc.
124:9026-9027; Chin, et al., (2002) Proc. Natl. Acad. Sci., 99:11020-11024;
Wang, et al., (2003) Proc. Natl.
Acad. Sci., 100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin, et al., (2003) Science, 300:964-967. This allows the selective labeling of virtually any protein with a host of reagents including ftuorophores, crosslinking agents, saccharide derivatives and cytotoxic molecules. See also, U.S.Patent Application Serial No. 10/686,944 entitled "Glycoprotein synthesis" filed January 16, 2003, which is incorporated by reference for the aforementioned disclosure. Post-translational modifications, including but not limited to, through an azido amino acid, can also made through the Staudinger ligation (including but not limited to, with triarylphosphine reagents). See, e.g.. Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modificatioir by the Stauditrger ligtation, PNAS 99(1):19-24.
IX. Alternate Systenrs For Producing Non-Natural Amino Acid Polypeptides 1004131 Several strategies have been employed to introduce non-natural ainino acids into proteins in non-recombinant host cells, mutagenized host cells,.or in cell-free systems.
Further information in this regard can be found in U.S. Patent Application No. 11/316,534.
X. Post-Translational Modifications of Non-Natural Amino Acid Components of a Polypeptide 1004141 For convenience, the post-translational modifications of non-natural amino acid components of a polypeptide described in this section (XA to XJ) have been described generically and/or with specific examples.
However, the post-translational modifications of non-natural amino acid components of a polypeptide described in this section should not be limited to just the generic descriptions or specific example provided in this section, but rather the post-translational modifications of non-natural amino acid components of a polypeptide described in this section apply equally well to all compounds that fall within the scope of Formulas I-XV and compounds having the structures 1-4, including any sub-formulas or specific compounds that fall within the scope of Forinulas I-XV and compounds having the structures 1-4, that are described in the specification, claims and figures herein.
1004151 Methods, compositions, techniques and strategies have been developed to site-specifically incorporate non-natural aniino acids during the in vivo translation of proteins. By incorporating a non-natural amino acid with a sidechain cheniistry that is orthogonal to those of the naturally-occurring amino acids, this technology allows the site-specific derivatization of recombinant proteins. As a result, a major advantage of the rimethods, compositions, techniques and strategies described herein is that derivatized proteins can now be prepared as defined homogeneous products. However, the methods, compositions, reaction mixtures, techniques and strategies described herein are not limited to non-natural aniino acid polypeptides formed by in vivo protein translation techniques, but includes non-natural amino acid polypeptides formed by any technique, including by way of example only expressed protein ligation, chemical synthesis, ribozyme-based techniques (see, e.g., section herein entitled "Expression in Alternate Systems").
1004161 The ability to incorporate non-natural amino acids into reconibinant proteins broadly expands the chemistries which may be implemented for post-translational derivatization, wherein such derivatization occurs either in vivo or in vitro. More specifically, polypeptide derivatization utilizing the reaction of a carbonyl and a hydrazine to an indole linkage on a non-natural aniino acid portion of a polypeptide offers several advantages.
First, the naturally occurring amino acids do not (a) contain carbonyl groups that can react with hydrazine groups to form indole linkage and (b) hydrazine groups that can react with carbonyl groups to form indole linkages, and thus reagents designed to form such linkages will react site-specifically with the non-natural amino acid component of the polypeptide (assuming of course that the non-natural, amino acid and the corresponding.reagent have been designed to form such a linkage), thus the ability to site-selectively derivatize proteins provides a single homogeneous product as opposed to the mixtures of derivatized proteins produced using documented methodologies. Second, such indole linkages are stable under biological conditions, suggesting that proteins derivatized by such indole linkages are valid candidates for therapeutic applications.
Third, the stability of the resulting indole linkage can be manipulated based on the identity (i.e., the functional groups and/or structure) of the non-natural amino acid to which the indole linkage has been formed. In some embodiments, the indole linkage to the non-natural amino acid polypeptide has a decomposition half life less than one hour, in other embodiments less than 1 day, in other embodiments less than 2 days, in other embodiments less than I week and in other embodiments more than I week. In yet other enibodiments, the resulting indole linkage is stable for at least two weeks under mildly acidic conditions, in other embodiments the resulting indole linkage is stable for at least 5 days under mildly acidic conditions. In other embodiments, the non-natural aniino acid polypeptide is stable for at least I day in a pH
between about 2 and about 8; in other embodiments, from a pH of about 2 to about 6; in other embodiment, in a pH of about 2 to about 4. In other embodiments,. using the strategies, methods, compositions and techniques described herein, an indole linkage to a non-natural amino acid polypeptide is synthesized with a decomposition half-life tuned to the situation at hand (e.g., for a therapeutic use such as sustained release, or a diagnostic use, or an industrial use or a military use).
1004171 The non-natural amino acid polypeptides described above are useful for, including but not linvted to, novel therapeutics, diagnostics, catalytic enzymes, industrial enzymes, binding proteins (including but not limited to, antibodies and antibody fragments), and including but not limited to, the study of protein structure and function. See; e.g., Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structetre and Function, Current Opinion in Cheniical Bioloev,, 4:645-652. Other uses for the non-natural amino acid polypeptides described above include, by way of example only, assay-based, cosmetic, plant biology, environmental, energy-production, and/or military uses. However, the non-natural amino acid polypeptides described above can undergo further modifications so as to incoiporate new or modified functionalities, including manipulating the therapeutic effectiveness of the polypeptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics, pharmacologics and/or pharmacodynamics of the polypeptide (e.g., increasing water solubility, bioavailability, increasing senim half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time), providing additional functionality to the polypeptide; incorporating a tag, label or detectable signal into the polypeptide, easing the isolation properties ofxhe polypeptide; and any combiiaation of the aforementioned modifications.
(00418.1 In certain 'embodiments are metliods for easing the isolation properties of a polypeptide comprising utilizing a homologous non-natural an-ino acid polypeptide comprising at least one, non-natural amino acid selected from the. group consisting _of a carbonyl-containing_ non-natural amino acid, a hydrazine-containing non-natural amino acid. In other embodiments such non-natural amino acids have been biosynthetically incorporated into the polypeptide. as described herein. In further or alternative embodiments such non-natural amino acid polypeptides comprise at least one non-natural amino acid selected froni amino acids of Formula I-XV. In further or alternative enibodiments such non-natural aniino acid polypeptides comprise at least one non-natural amino acid selected from amino acids of compounds having structures 1-4.
1004191 The methods, compositions, strategies and techniques described herein are not limited to a particular type, class or farnily of polypeptides. Virtually any polypeptide may include at least one non-natural amino acids described herein. By way of example only, the polypeptide can be homologous to a therapeutic protein.
'rhe non-natural amino acid polypeptide may also be homologous to any polypeptide member of the growth hormone supergene family.
[004201 Such modifications include the incorporation of fuztlter' functionality onto the non-natural amino acid component of the polypeptide, including but not.limited to, a desired functionality.
1004211 In addition, non-natural amino acid polypeptides may contain moieties which may be converted into other functional groups, such as, by way of exantiple only, carbonyls or hydrazines. FIGS. 23 illustrates the chemical conversion of non-natural amino acid polypeptides into carbonyl-containiiig non-natural amino acid polypeptides and hydrazine containing non-natural an-dno acid polypeptides.
The resulting hydrazine- and carbonyl-containing non-natural amino acid polypeptides may be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing, and using non-natural aniino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. The chcmical conversion of chemical moieties into other functional groups, such as, by way of example only, carbonyls, or hydrazinc can be achieved using documented methodologies, such as described, for example, in March, ADVANCED ORGANIC CHEMISTRY 5`s Ed., (Wiley 2001); and Carey and Sundberg, ADVANCED
ORGANIC CHEMISTRY 4th Ed., Vols. A and B (Plenum 2000, 2001).
1004221 Furthermore, the chemical modification of carbonyl-containing non-natural amino acid polypeptides with hydrazine containing reagents can be used to generate highly fluorescent indole derivative containing non-natural amino acid polypeptides under the appropriate excitation. Fig. 19 and 21 illustrate the chemical modification of carbonyl containing non-natural amino acid polypeptides with hydrazine containing reagents. In addition, hydrazine containing non-natural anzino acid polypeptides can chemically react with carbonyl containing reagents to form highly fluorescent. indole derivative containing non-natural amino acid poypeptides under the appropriate excitation. Fig. 20 and 22 illustrate the chemical modification of hydrazine containing non-natural amino aeidpolypeptide with carbonyl containing reagents.

A -Methods for Post-Translationally Modifying Non-Natural Amino Acid Polypeptides:
Syntl:esis of indole-containing non-natural amino acid polypeptides 1004231 The incorporation of substituted carbonyl and substituted hydrazine-containing non-natural aniino acids to polypeptides provides the site-specific derivatization via the forrnation of an indole linkage. The methods for derivatizing and/or further rriodifying may be conducted with a polypeptide that has been purified prior.to -the derivatization step or after the derivatization step. In addition, the methods for derivatizing and/or further modifying 'may be conducted with synthetic polymers, polysaccharides, or polynucleotides which have been purified before or after such modifications. In addition, derivatization step can occur efficiently under mildly acidic,. including by way of example, between a pH of about Ito about 6.
[004241 Furthermore, certain indole linkages allow the production of fluorescent non-natural. amino acid polypeptides that are used in a variety of detection methods.
1004251 Fig. 21 illustrates the site specific labeling of carbonyl containing non-natural amino acid polypeptides with hydrazine containing reagents. Fig. 22 illustrates the site specific labeling of hydrazine containing non-natural amino acid polypeptides with carbonyl containing reagents:
1004261 In addition, the derivatization may be performed using reagents containing carbonyl or hydrazine groups on one end and functional groups on the other. The resulting indole-containing non-natural amino acid.
polypeptides can be further modified to introduce molecules, including by a way of example only polymers, polysaccharides, or polynucleotides. Fig. 24B represents illustrative, non-limiting examples of the reaction of functional group containing polypeptides with PEG derivatives.
1004271 By way of example only, the reagents of formula (XVI) are the type of carbonyl- or hydrazine-containing reagents that can be used to form indole-containing non-natural aniino acid polypeptides and can further be modified to introduce other molecules. In one embodiment, hydrazine-containing compounds of Formula I to IV can react with reagent of Formula XVI containing carbonyl group to form indole-containing.
non-natural amino acid polypeptides. In another embodiment, carbonyl-containing compound of Formula V to XIV can react with reagent of Formula XVI containing hydrazine group to form indole-containing non-natural amino acid polypeptides.
~X L L, W
(XVI) wherein:
each X is independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")Z, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R", -C(O)ZR", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl;
or each X is independently a desired functionality;
eacli L is independently selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)NTR'C(O)O-(alkylene or substituted alkylene)-, -O-CON(R'){alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -N(R')C(O)O-(alkylene or substituted alkylene)-, -S(O)kN(R')7, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-(alkylene or substituted alkylene)-, -N(R')C(S)N(R')-, -N(R')S(O)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')Z-N=N-, and -C(R')z-N(R=')-N(R')-;
Li is optional, and when present, is -C(R')P NR'-C(O)O-(alkylene or substituted alkylene)- where p is 0, 1, or 2;
each R' is independently H, alkyl, or substituted alkyl;
R"rO H
~! \ N`NHZ
W is ~- , and R is H. alkyl, or substituted alkyl;
1004281 In certain einbodiments of compounds of Formula (XVI), X is a polymer comprising alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl. In certain embodiinents of conipounds of Fomiula (XVI), X is a polymer comprising polyalkylene oxide or substituted polyalkylene oxide. In certain embodiments of conipounds of Formula (XVI), X
is a polymer coniprising -[(alkylene or substituted alkylene)-O-( hydrogen, alkyl, or substituted alkyl)],x, wherein x is from about 20 to about 10,000. In certain embodiments of compounds of Formula (XVI), X is m-PEG
having a molecular weight ranging from about 2 to about 40 KDa. In certain embodiments of compounds of Formula (XVI), X is a biologically active agent selected from the group consisting of a peptide, protein, enzyme, antibody, drug, dye, lipid, nucleoside, oligonucleotide, cell, virus, liposonie, microparticle, and micelle. In certain embodiments of compounds of Formula (XVI), X is a drug selected from the group consisting of an andbiotic, fungicide, anti-viral agent, anti-inflamrnatory agent, anti-tumor agent, cardiovascular agent, anti-anxiety agent, hormone, growth factor, and steroidal agent. In certain embodiments of compounds of Formula (XVI), X is an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, 0-galactosidase, and glucose oxidase. In certain embodiments of compotuids of Formula (XVI),, X is a detectable label selected from the group consisting of a fluorescent, phosphorescent, chemiluminescent, chelating, electron dense, magnetic, intercalating, radioactive, chromophoric, and energy: transfer moiety. In certain embodiments of compounds of Formula (XVI), X is a reactive group consisting of carbonyl containing moiety and hydrazine containing moiety. In certain embodiments of compounds of Formula (XVI), X is a indole derivatives. In certain embodiments of compounds of Formula (XVI), L is selected froni the.group consisting of -N(R')CO-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -N(R')C(O)N(R')-(alkylene or substi(uted alkylene)-, -O-CON(R')-(alkylene or substituted alkylene)-, -O-(alkylene or substituted alkylene)-, -C(O)N(R')-, and -N(R')C(O)O-(alkylene or substituted alkylene)-.
1004291 In certain enibodiments of compounds of Formula (XVI), are compounds having the structure of Formula (XVTI):
X-L-W (XVII) wherein:
H
R` O NH2 W is - , and ;_R is H, alkyl, or substituted alkyl.
1004301 In certain embodiments of compounds of formula (XVII), are compounds having the structure of Formula (XVIII):
H
" / N`NHZ O
~-o ~,'N~`' 0 m-PEG N N ~ I m-PEG
H
/ N`NH2 m-PEG N ~ ~ m-PEG N~/~/
O O OI and H O
I m-PEG OuN~~O~
0 ; where in other embodiments such m-PEG or PEG groups have a molecular weight ranging from about 5 to about 30 kDa.
1004311 In certain embodiments of compounds of Formula (XVII), are compounds having the stnicture of Formula (XIX):
x ~ O
NJ" O'~Y\W
X H
(XIX).
wlierein:
H
N

I
W is , and ~ ; R is H, alkyl, or substituted alkyl.
Y when present is alkyl, or substituted alkyl.
L is -(alkylene or substituted alkylene)-N(R')C(O)O-(alkylene or substituted alkylene)-. In certain embodiments of conipounds of Formula (XVII), are compounds having the structure of Formula (XX):

Im-P EG

L O
~N"J~O"'N~\W
H m-PEG L

(XX).
wherein other embodiments of compounds of Formula (XX) such m-PEG groups have a molecular weight ranging from about 5 to about 30 kDa.

11.8 (00432) In certain embodiments of compounds of Formula (XVII), are compounds having the structure of Formula (XXI):
x ~ O
X-L
L'~"J' N O/ W
X H
(XXI).
wherein:
H
R O 0(NH2 W is , and ; R is H, alkyl, or substituted alkyl.
Y when is present is alkyl, or substituted alkyl.
L is -(alkylene or substituted alkylene)-N(R')C(O)O-(alkylene or substituted alkylene)-. In certain embodiments of compounds of Formula (XXI), are compounds having the stnicture of Formula (XXII):

m-PEG

Lm-PEG L H O/Y\ W
L
I
m-PEG
(XXII).
wherein other embodiments of compounds of Fomiula (XXII) such m-PEG groups have a molecular weight ranging from about 5 to about 30 kDa.
1004331 In certain embodiments, linkers of Formula (XVII) are reactive with carbonyl- or hydrazine-containing polypeptide in aqueous solution under acidic conditions. In certain embodiments, such acidic conditions are pH about 1 to about 6.
1004341 In ccrtain embodiments of compounds of Formula (XVII), are conipounds having the structure of Formula (XXIII):
O O
W Z O"J Z W
(XXIII) wherein:
ZisOorNHandnis 1,2,3and4 H
~ , NHZ
R'T '0 N
W is and R is H, alkyl, or substituted alkyl. ln certain embodiments of compounds.of Formula (XXIII), are compounds having the structure of Fomiula (XXIV):
O O
eN-__~0_1~04n ~/
ON I HyN~ ~ /NHZ
H H
(XXIV) 1004351 In other embodiments of compounds of Formula (XXIII), are compounds having the structure of Formula (XXV) p O
H/ v O

(XXV) 1004361 In certain embodiments are methods for derivatizing a polypeptide comprising amino acids of Formulas I-XV and compounds having the structures 1-4, including any sub-formulas or specific compounds that fall within the scope of Formulas I-XV, wherein the method comprises contacting the polypeptide comprising at least one amino acid of Formula I-XV with a reagent of Formula (XVI). In certain embodiments the polypeptide is purified prior to or after contact with the reagent of Formula (XVI). In other embodiments are resulting polypeptide coniprises at least one carbonyl- or one hydrazine-containing amino acid of formula I-XV.
In other embodiments are resulting polypeptide comprises at least one indole-containing polypeptide generated from the coupling of compounds of formula I-XV with the reagent of formula (XVI).
(004371 Figure 26 provides an illustrative example of the synthesis of bifunctional linker of formula (XXIV). Wherrin the method comprises coupling a spacer reagent containing on both ends an amine or hydroxyl group to acid containing Boc-protected hydrazine. The cleavage of Boc:group leds to linkers of formula (XXIV).
1004381 Fig 27 provides a schematic representation of post-translational modification of polypeptide containing carbonyl non-natural amino acid with reagent of formula (XXIV) to form indole containing polypeptide.
1004391 Fig 28 provides a scheniatic representation of post-translational modification of polypeptide containing carbonyl non-natural amino acid with reagent of formula XX and XXII
to form indole containing polypeptide.
1004401 Fig 29 provides a schematic representation of post-translational modification of polypeptide containing hydrazine non-natural amino acid with reagent of formula XX and XXII to form indole containing polypeptide.

1004411 Fig 30 illtistrates examples of reagents of formula (XVIII).
1004421 In certain embodiments are methods for producing a polypeptide dimer via indole linkage, wherein the method consists of the reaction of a linker of formula (XXIII) with carbonyl- or hydrazine-containing non-natural amino acid polypeptide. Fig. 27 provides a representative ezample of the formation of such dimmer using condensation of linker of formula (XXIV) with carbonyl-containing non-natural amino, acid polypeptide.
(00443] In certain embodiments are methods for preparing a polypeptides containing an indole moiety via the use of bifunctional linker, wherein the metliod comprises:
(i) derivatizing a first polypeptide comprising an atnino acid of Formula.(1) with a bifunctional linker, and (ii) contacting the resulting_derivatized protein of step (i) with a second second reagent, such as;PEG. In certain embodiments the polypeptides are purified prior to or after contact with the bifunctional linker.
1004441 Fig. 24 shows a illustrative euample of such bifunctional linker. and its use to produce indole containing polypeptide attached to PEG group.
1004451 By way of example only, the following are representative examples of bifnnctional linkcrs of formula (XXVI).

SR 0~S~ S-3 W-L-O-NHZ W-L~ W-L-/ W--L--f Ar (XXVI) Wherein;
H
N
R~O i ~ ~NH2 W is ^^^- , and R is H, alkyl, or substituted alkyl; and L is -(alkylene orsubstituted alkylene)=N(R')C(O)O-(alkylene,or substituted alkylene)-.
(00446) In one embodiment, multiple linker ehemistries can react site-specifically with a carbonyl- or a hydrazine-contaitiing non-natural amino acid polypeptide. In one embodiment,, the linker methods described herein utilize linkers containing the hydrazine functionality on at least one liiilcer terinini (mono, bi- or multi-functional). The, reaction of an hydrazine-derivatized linker with a carbonyl-substituted protein generates an indole substituted non-natural protein. ln other embodiments,, the linker methods described herein utilize linkers containing the carbonyl functionalityon at least one linker termini (mono, bi-or multi-functional). The reaction of carbonyl-derivatized linker with a hydrazine-substituted protein generates an indole substituted non-natural protein.
100447E In certain embodiments are methods for derivatizing a chemically synthesized polypeptide comprising carbonyl- or hydrazine-containing non-natural polypeptide with carbonyl or hydrazine containing reagents to form indole derivatives.
1004481 Figure 19 provides illustrative exaniples of the derivatization of carbonyl-containing Urotensin with hydrazine containing reagents. In this illustrative embodiment, hydrazine-containing reagents are added to a buffered solution (pH 1-5) of carbonyl-containing Urotensin analogs. The reaction proceeds at ambient teinperature for hours to days.

l21 1004491 Figure 20 provides illustrative exainples of the derivatization of hydrazine-containing Urotensin with carbonyl containing reagents. In this illustrative embodiment, carbonyl-containing reagents are added to a buffered solution (pH 1-5) of hydrazine-containing Urotensin analogs. The reaction proceeds at ambient temperature for hours to days.
1004501 In other embodiments such derivatized polypeptides are stable in aqueous solution for at least 1 month under mildly acidic conditions. In other embodiments such derivatized.polypeptides are stable for at least 2 weeks under mildly acidic conditions. In other embodiments such derivatized polypeptides are stable for at least 5 days under mildly acidic conditions. In other embodiments.such conditions are pH about 1 to about 6. In certain embodiments the tertiary structure of the.derivatized potypeptide is preserved. In other embodiments such derivatization of polypeptides further comprises ligating the derivatized polypeptide to another polypeptide. Iri-other embodiments stich polypeptides being derivatized are homologous to a therapeutic protein.
B. E.ranrple of Adding Functionalily: Macron:olecular Polynters Coupled to Non-Natural Amino Acid Pulypeptides 1004511 Various modifications to the non-natural amino acid polypeptides described herein can be effected using the compositions, methods, techniques andstrategies described herein.
These modifications include the incorporation of further functionality onto the non-natural amino acid component of the polypeptide, including but not limited to, a desired functionality. As an illustrative, non-limiting example of the compositions, methods, techniques and strategies described herein, the following description will focus on adding macromolecular polymers to the non-natural amino acid polypeptide with the understanding that the compositions, methods, techniques and strategies described thereto are also applicable to adding other functionalities, including but not limited to those listed above.
1004521 A wide variety of macromolecular polymers and other moleculcs can be coupled to the non-natural amino acid polypeptides described hercin to modulate biological properties of the non-natural amino acid polypeptide (or the corresponding natural amino acid polypeptide), and/or provide new biological properties to the non-natural arriino acid polypeptide (or the corresponding natural amino, acid polypeptide). These macromolecular polymers can be coupled to the non-natural amino acid polypeptide via_ the non-natural amino acid, or any functional substituent of the non-natural amino acid, or any substituent or fimctional group added to the non-natural amino acid.
1004531 Water soluble polymers can be coupled to the non-natu.ral amino acids incorporated into polypeptides (natural or synthetic), polynucleotides, poly saccharides or synthetic polymers described herein. The water soluble polymers may be coupled via a-non-natural amino acid incorporated in the polypeptide or any functional group or substituent of a non-natural amino acid, or any functional group or substituent added to a non-natural amino acid. ln some cases, the-non-natural amino acid polypeptides described herein comprise one or more non-natural amino acid(s) coupled to water soluble polymers and one or more naturally-occurring amino acids linked to water soluble polyniers. Covalent attachment of hydrophilic polymers to a biologically active molecule represents one approach to increasing water solubility (such as in a physiological environment), bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time of the biologically active molecule, including proteins, peptides, and particularly hydrophobic molecules. Additional important features of such hydrophilic polymers include biocompatibility, lack of toxicity, and lack of immunogenicity. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable.

[004541 Examples of hydrophilic polyniers include, but are not limited to:
polyalkyl ethers and'alkoxy-capped analogs thereof (e.g:, polyoxyethylene glycol, =polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogs thereof, especially polyoxyethylene glycol, the latter is also known as polyethylene glycol or PEG); polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl.acrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropylmethacrylamide and derivatives thereof); polyliydroxyalkyl acrylates; polysialic acids and analogs thereof; hydrophilic peptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives, e.g., carboxymethyldextran, deztran sulfates, aminodextran; cellulose and its derivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses; chitin and its derivatives, e.g., chitosan, succinyl chitosan, carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and its derivatives; starches; alginates;
chondroitin sulfate; albumin; pullulan and carboxvmethyl pullulan;
polyaminoacids and derivatives thereof, e.g., polyglutamic acids, polylysines, polyaspartic acids, polyaspartamides; maleic anhydride copolymers such as:
styrene nialeic anhydride copolymer, divinylethyl ether maleic anhydride copolymer; polyvinyl alcohols;
copolymers thereof,=, terpolymers thereof; mixtures thereof; and derivatives of the foregoing. The water soluble polymer may be any structural form including but not.limited to linear, forked or branched. In. sonie embodiments, polymer backbones'that are water-soluble, with from 2 to about.
300 termini, are particularly useful. Multifunctional polymer derivatives include, buk,are not limited to, linear polyn-ers having two termini, each terminus being bonded to a functional group which may be the same or different: In soineembodiments, the water polymer comprises a poly(ethylene :glycol) moiety. The molecular weight of the polymer may be within a desired polymer:molecular weight range. The foregoing list for substantially water soluble backbones is by no means exhaustive and is merely illustrative, and that all polymeric materials having the qualities described above are contemplated as being suitable for use in methods and compositions described herein.
1004551 As described above, one exaniple of a hydrophilic polymer is polyethylene glycol, abbreviated PEG, which has been used extensively in pharmaceuticals, on artificial implants, and in other applications where biocompatibility, lack of toxicity, and lack of inununogenicity are of iniportance. The polymer:polyeptide embodiments described herein will use PEG as an example hydrophilic polymer with the understanding that other hydrophilic polymers may be siniilarly utilized in such embodiments.
1004561 PEG is water soluble polymer that is conunercially available or can be prepared by ring-opening polymerization of ethylene glycol according to documented methodologies (Sandier and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). PEG is typically clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze or deteriorate, and is generally non-toxic. Poly(ethylene..=glycol) is considered to be biocompatible; which is to.say that PEG is capable of :coexistence, with. living tissues or organisms without causing harm. More specifically, PEG is substantially non-immunogenic, which i's tosay that PEG does not tend to produce an immune response.in the body. When attached to a molecule having some desirable function in the body, such as a biologically active agent; the PEG tends to mask the agent and can reduce or eliminate any immune.responseso that an organism can tolerate the presence of the agent. PEG conjugates tend not to produce a substantial. immune response or cause clotting or other undesirable effects.

[004571 The term "PEG" is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented as linked to a non-natural amino acid polypeptide by the formula:
XO-(CHZCHzO)õ-CHZCH2-Y
where, n is 2 to 10,000 arid X is H or a terminal modification, including but not Gmited to, a C1.4 alkyl, a protecting group, or a terminal functional group. The. term PEG includes, but is not limited to, polyethylene glycol in any of its forms, including bifunctional PEG, multiarmed PEG, derivatized PEG, forked PEG, branched PEG (with each chain having a molecular weight of from about 1 kDa to about 100 kDa, from about I
kDa to about 50 kDa, or from about 1 kDa to about 20 kDa), pendent PEG (i.e.
PEG or related polymers having one or more functional groups pendent to the polymer backbone), or PEG with degradable linkages therein. In one embodiment, PEG in which n is from about 20 to about 2000 is suitable for use in the methods and compositions described herein. In some embodiments, the water polymer comprises a polyethylene glycol moiety. The molecular weight of the PEG polynier may be of a wide range including but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weiglit of the PEG
polymer may be within a desired polymer molecular weight range. A wide range of PEG molecules are described in, including but not limited to, the Shearwater Polymers, Inc. catalog, Nektar Therapeutics catalog, incorporated herein by reference.
1004581 Specific examples of terminal functional groups in the literature include, but are not limited to, N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698, 5,468,478), anune (see, e.g., Buckmann et al.
Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (Sec, e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succininiidyl propionate and succinimidyl butanoate (see, e:g., Olson et al. inPoly(ethylene glycol) Cliemistry & Biological Applications, pp 170-18 1, Harris & Zalipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.. No. 5,672,662), succinimidyl succinate (See, e:g., Abuchowski et al. Cancer Biochem. Biophys. 7:_175 (1984) and Joppich et al.
Makromol. Chem. 180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.
5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur..J Biochem. 94:11 (1979), Elling et al., Biotech. Appl.
Biochem. 13:354 (1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.
Biochem: Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No.
5,824,784, U.S. Pat. No. 5,252,714), nialeimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romani et al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan, Synthetic Conun. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of the above references and patents. are incorporated herein by reference in their entirety.
1004591 In some cases, a PEG terminates on one end with hydroxy or metlioxy, i.e., X is H or CH3 ("methoxy PEG"). Alternatively, the PEG can terminate with a reactive group, thereby forming a bifunctional polynier.
Typical reactive groups can include those reactive groups that are commonly used to react with the functional groups found in the 20 common amino acids (including but not limited to, maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester and aldehydes) as well as functional groups that are inert to the 20 1.24 conunon amino acids but that react specifically with complementary functional groups present in non-natural amino acids (including but not liniited to, phenyl hydrazine and carbonyl groups).
1004601 It is noted that the other end of the PEG, which is shown in the above formula by Y, will attach either directly or indirectly to a polypeptide (synthetic or natural), polynucleotide, polysaccharide or synthetic polymer via a non-natural amino acid. When Y is a phenyl liydrazine group, then the phenyl hydrazine-containing PEG
reagent can react with a carbonyl-containing non-natural amino acid in a polypeptide to form a PEG group linked to the polypepride via an indole linkage. When Y is a carbonyl group, then the carbonyl-containing PEG
reagent can react with a phenyl hydrazine-containing non-natural amino acid in a polypeptide to form a PEG
group linked to the polypeptide via an indole linkage: FIG. 30 presents non-limiting examples carbonyl- and hydrazine-containing PEG reagents.
1004611 In some embodiments, a hydrazine can be reacted with a carbonyl group present in a non-natural amino acid to form an indole. Alternatively, the hydrazine can be incorporated into the polypeptide via a non-natural amino acid and used to react preferentially with a carbonyl group present in the water soluble polymer.
Generally, at least one terminus of the PEG molecule is available for reaction with the non-natural amino acid.
1004621 Thus, in some embodiments, the polypeptide comprising the non-natural amino acid is linked to a water soluble polymer, such as polyethylene glycol (PEG), via the side chain of the non-nattiral aniino acid. The non-natural amino acid methods and compositions described herein provide a highly efficient method for the selective modification of proteins with PEG derivatives, which involves the selective incorporation of non-natural amino acids, including but not limited to, those amino acids containing functional groups or substituents not found in the 20 naturally incorporated ainino acids, into proteins in response to a selector codon and the subsequent modification of those amino acids with a suitably reactive PEG
derivative. Documented methodologies of a wide variety are suitable for use with the non-natural amino acid methods and compositions descnbed herein to incorporate a water soluble polymer into the protein.
1004631 The polymer backbone can be linear or branched. Branched polymer backbones have been generally documented. Typically, a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core. PEG is used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. The central branch moiety can also be derived from several amino acids, such as lysine.
The branched poly(ethylene glycol) can be represented in general form as R(-PEG-OH)m in which R is derived from a core moiety, such as glycerol, glycerol oligomers, or pentaerythritol, and m represents the nuniber of arms.
Multi-armed PEG molecules, such as ttiose described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490;
4,289,872; U.S. Pat. Appl. 2003/0143596;
WO 96/21469; and WO 93/21259, each of which is incorporated by reference herein for the aforementioned disclosure, can also be used as the polymer backbone.
1004641 Branched PEG can also be in the form of a forked PEG represented by PEG(-YCHZ,),,, where Y is a linking group and Z is an activated terminal group linked to CH by a chain of atoms of defined length. Yet another branched form, the pendant PEG, has reactive groups, such as carboxyl, along the PEG backbone rather than at the end of PEG chains.
1004651 In addition to these forms of PEG, the polymer can also be prepared with weak or degradable linkages in the backbone. For example, PEG can be prepared with ester linkages in the polymer backbone that are subject to liydrolysis. As shown herein, this hydrolysis results in cleavage of the polynier into fragments of lower molecular weight:

-PEG-COZ-PEG-+HZO 4 PEG-CO2H+HO-PEG-The term polyethylene glycol or PEG represents or includes all documented fornis including but not limited to those disclosed herein. The niolecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer may be within a desired polymer molecular weight range.
1004661 In order to nuzxintize the desired properties of PEG, the total molecular weight and hydration state of the PEG polyiner or polymers attached to the biologically active molecule must be sufficiently high to impart the advantageous characteristics typically associated with PEG polymer attachment, such as increased water solubility and circulating half life, while not adversely impacting the bioactivity of the parent molecule.
(004671 The methods and compositions described herein may be used to produce substantially homogenous preparations of polymer:protein conjugates. "Substantially honiogenous" as used herein means that polymer:protein conjugate molecules arc observed to be greater than half of the total protein. The polymer:protein conjugate has biological activity and the present "substantially homogenous" PEGylated polypeptide preparations provided herein are those which are homogenous enough to display the advantages of a homogenous preparation, e.g., ease in clinical application in predictability of lot to lot pharmacokinetics.
1004681 One may also choose to prepare a mixture of polymei=:protein conjugate molecules, and the advantage provided herein is that one may select the proportion of mono-polymer:protein conjugate to include in the mixture. Thus, if desired, one may prepare a mixture of various proteins with various numbers of polymer moieties attached (i.e., di-, tri-, tetra-, etc.) and combine said conjugates with the mono-polymer:protein conjugate prepared using the methods described herein, and have a mixture with a predeternvned proportion of mono-polymer:protein conjugates.
1004691 The proportion of polyethylene glycol molecules to protein molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is minimal excess unreacted protein or polymer) may be determined by the molecular weight of the polyethylene glycol selected and on the number of available reactive groups available. As relates to molecular weight, typically the higher the molecular weight of the polymer, the fewer number of polymer molecules which niay be attached to the protein. Sirnilarly, branching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polymer:protcin ratio.
1004701 As used herein, and when contemplating hydrophilic polymer:polypeptide/protein conjugates, the term "therapeutically effective amount" further refers to an amount which gives an increase in desired benefit to a patient. The amount will vary from one individual to another and will depend upon a number of factors, including the' overall physical condition of the patient and the underlying cause of the disease, disorder or condition to be treated.
1004711 The number of water soluble polymers linked to a modified or unmodified non-natural amino acid polypeptide (i.e., the extent of PEGylation or glycosylation) described herein can be adjusted to provide an altered (including but not limited to, increased or decreased) pharmacologic, pharmacokineuc or pharmacodynamic characteristic such as in vivo half-life. In some embodiments, the half-life of the polypeptide is increased at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, two fold, five-fold, 10-fold, 50-fold, or at least about 100-fold over an unmodified polypeptide.

1004721 In one embodiment, a polypeptide comprising a carbonyl-contaiiiing non-natural amino acid is modified with a PEG derivative that contains a terminal arylhydrazine moiety that is linked directly to the PEG
backbone.
1004731 In one embodiment, a polypeptide comprising a carbonyl-containing non-natural amino acid is modified with a PEG derivative that contains-a terminal hydrazine moiety that is linked directly to the PEG
backbone.
1004741 In another embodinient, a polypeptide comprising a hydrazine-containing amino acid is modified with a PEG derivative that contains a terminal carbonyl moiety that is linked directly to the PEG
backbone.
1004751 In some.embodiments, the carbonyl-terminal PEG derivatives have the structure:
RO-(CHZCH2O),,-0-(CHZ)Z-NH-C(O)(CH2),,; C(O)-R
where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (i.e., average molecular weight is between 5-40 kDa). The molecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer may be within a desired polymer molecular weight range.
(004761 Several reviews and monographs on the functionalization and conjugation of PEG are available. See, for example, Harris, Macromol. Chem. Pliys. C25: 325-373 (1985); Scouten, Methods in Enzyrnology 135: 30-65 (1987); Wong et, al., Ettzyme Microb. Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);
Zalipsky, Bioconjugate Chem. 6:
150-165 (1995).
1004771 Methods for activation of polymers can also be found. in WO 94/17039, U.S. Pat. No.
5,324,844õ WO 94/18247, WO 94/04193, U.S. Pat. No. 5;219,564; U.S. Pat. No.
5,122,614, WO 90/13540, U.S.
Pat. No. 5,281,698, and more WO 93/15189, and for conjugation between activated polymers and enzymes including but, not limited to Coagulation Factor VIII (WO 94/15625), haemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.
Biochein. Biotech. 11: 141-152 (1985)), all of which are herein incorporated by reference for the aforementioned disclosure.
1004781 If necessary, the PEGylated non-natural amino acid polypeptides described herein obtained from the hydrophobic chroina.tography can be purified.further by documented methodologies including, but are not limited to, affinity chromatography; anion- or cation-exchange chromatography (using, including but not limited to, DEAE SEPHAROSE); chromatography on silica; reverse phase HPLC; gel filtration (using, including but not limited to, SEPHADEX G-75); hydrophobic interaction chromatography; size-exclusion chromatography, metal-chelate chromatography; ultrafiltration/diafiltration;
ethanol precipitation; ammonium sulfate precipitation; cliromatofocusing; displacement chromatography;
electrophoretic procedures (including but not limited to preparative isoelectric focusing), differential solubility (including but not limited to ammonium sulfate precipitation), or extraction. Apparent niolecular weight may be estimated by GPC by coinparison to globular protein standards (Preneta AZ, PROTEIN PURIFICATION
METHODS, A PRACTICAL
APPROACfi (Harris & Angal, Eds.) IRL Press 1989,, 293-306). The purity of the (non-natural amino acid polypeptide):PEG conjugate can be assessed by proteolytic degradation (including but not limited to, trypsin cleavage) followed by niass spectrometry analysis. Pepinsky R.B., et.al., J.
Pharmacal. & Exp. Ther.
297(3):1059-66(2001).

[004791 A water soluble polymer linked to a non-natural amino acid of a polypeptide described herein can be further derivatized or substituted without limitation.

C. Enbanci-ig affinityfor seruni albumin (00480) Various molecules can also be fused to the non-natural amino acid polypeptides described herein to modulate the half-life in serurn In some embodiments, molecules are linked or fused to the modified or unmodified non-natural amino acid polypeptid8s describ"ed herein to enhance affinity for endogenous serum albumin in an aninial.
1004811 For example, in some cases, a recombinant fusion of a polypeptide and an albumin binding sequence is made. Exemplary albumin binding.sequences include, but are not limited to, the albumin binding domain from streptococcal protein G(see. e.g., Makrides et al., J. Pharmacol.
Exp. Ther. .277(1):534-542 (1996) and Sjolander el al., J, /mmunol. Methods 201:115-123 (1997)), or albumin-binding peptides such as those described in, e.g., Dennis, et al., J. Biol. Chem. 277(38):35035-35043 (2002).
(00482) In other embodiments, the modified or unmodified non-natural amino acid polypeptides described herein are acylated with fatty acids. In some cases, the fatty acids promote binding to serum albumin.
See, e.g., Kurtzhals, et al., Biochem. J. 312:725-731 (1995).
1004831 In other embodiments, the modified or unmodified non-natural amino acid polypeptides described herein are fused directly with serum albumin (including but not limited to, human serum albumin). A
wide variety of other niolecules can also be linked to non-natural arimino acid polypeptides, modified or unmodified, as described herein, to modulate binding to serum albumin or other serum components.

A. Glycosylation of non-natural aniino acid polypeptides described Irerein 100484j The methods and compositions described herein include polypeptides incorporating one or niore non-natural amino acids bearing saccharide residues. The saccharide residues may be either natural (including but not limited to, N-acetylglucosamine) or non-natural (including but not limited to, 3-fluorogalactose). The saccharides may be linked to the non-natural,amino acids either by an N- or 0-linked glycosidic linkage (including but not limited to, N-acetylgalactose-L-serine) or a non-natural linkage (including but not lirnited to, a heterocycle, including a nitrogen-containing heterocycle, linkage or the corresponding C- or S-Iinked glycoside).
(00485) The saccharide (including but not limited to, glycosyl) moieties can be added to the non-natural amino acid polypeptides either in vivo or in vitro. In some embodiments, "a polypeptide comprising a carbonyl-containing non-natural amino acid is. modified with a saccharide derivatized with an arylhydrazine group to generate the corresponding glycosylated polypeptide linked via an indole linkage. In other enibodiments, a polypeptide comprising an arylhydrazine-containing non-natural amino acid is modified with a saccharide derivatized with a carbonyl group to generate. the corresponding glycosylated polypeptide linked via a linkage. Once attached to the non-natural amino acid, the saccharide may be further elaborated by treatment with glycosyltransferases and other enzymes to generate an oligosaccharide bound to the non-natural amino acid polypeptide. See, e.g., H. Liu, et al. J. Ani. Client. Soc. 125: 1702-1703 (2003).
1004861 FIG. 26 presents an illustrative example of the synthesis of a bifunctional homolinker in which the linker has two identical ends, i.e., hydrazine groups. Such a linker may be used to form a homodimer o1' a carbonyl-containing non-natural amino acid polypeptide to form two indole linkages. Alternatively, if one end of such a linker is protected, then such a partially protected linker can be used to bind the unprotected hydrazine end to a carbonyl-containing non-natural aniino acid polypeptide via an indole linkage, leaving the other protected end available for further linking reactions following deprotection. Alternatively, careful manipulation of the stoichiometry of the reagents inay provide a similar result (a heterodirner), albeit a result in which the desired heterodimer will likely be contaminated with some homodimer.
1004871 FIG. 27 presents an illustrative example of protein dimerization by coupling two proteins via a b'-functional homolinker.
[004881 FIG. 25 presents an illustrative example of the use of a heterobifunctional linker in which the linker has two different cnds, by way of example only a carbonyl group and a reactive group. In addition, FIG. 25. presents illustrative example of the use of a heterobifunctional linker to attach a PEG
group to a non-natural amino acid polypeptide in a multi-step synthesis. In the first step, as depicted in this illustrative figure, a hydrazine-containing non-natural amino acid polypeptide reacts with a carbonyl-containing bifunctional linker to form an indole-containing non-natural an-tino acid polypeptide. However, the bifunctional linker still retains a reactive functional group which reacts in a second step with a PEG reagent to form a~PEGylated non-natural amino acid polypeptide via a heterocycle linkage.
1004891 The methods and compositions described herein also provide for polypeptide combinations, such as homodimers, heterodimers, homomultimers, or. heteromultimers (i.e., trimers, tetramers,, etc:). By way of example only, the follovving description focuses on the- GH supergene family members, however, the methods, techniques and compositions described in this section can be applied to virtually any other polypeptide which can provide benefit iri the form of dimers and multimers, including by way of example only a therapeutic protein.
1004901 Thus, encompassed within the methods, techniques and coanpositions described herein are a GH supergene family member polypeptide containing one or more non-natural amino acids bound to another GH supergene family member or variant thereof or any other polypeptide that is a non-GH supergene family mcmber or variant thereof, either directly to the polypeptide backbone or via a linker. Due to its increased molecular weight conipared to monomers, the GH supergene family-member dimer or multimer conjugates may exhibit new or desirable properties, including but not limited to different pharmacological, pharmacokinetic, pharmacodynamic, modulated therapeutic half-life, or modulated plasma half-life relative to the monomeric GH
supergene family nlember: In some embodiments, the GH supergene family meniber dimers described herein will modulate the dimerization of the GH supergene family member receptor. In other embodiments, the GH
supergene family member dimers or multimers described herein will -act as a GH
supergene family member receptor aritagonist; agonist; or modulator.
1004911 In some embodiments, the- GH supergene family member polypeptides are linked directly, including but not:limited to, via an Asn-Lys.amide linkage or Cys-Cys disulfide linkage. In some embodiments, the linked GH supergene family member polypeptides, and/or the linked non-GH
supergene family member, will 'comprise different non-natural `amino acids to facilitate dimerization, including but not limited to, a first GH supergene family member,,and/or the linked.non-GH supergene family member, polypeptide comprising a carbonyl-containing- non-natural amino acid conjugated to a second GH
supergene family member polypeptide comprising a hydrazine-containing non-natural amino acid and the polypeptides are reacted via formation of the corresponding indole.

1004921 Alternatively, the two GH supergene fanuly member polypeptides, and/or the linked non-GH
supergenc family member, are linked via a linker. Any hetero- or homo-bifunctional linker can be used to link the two GH supeigene family member, and/or the linked non-GH supergene family meniber, polypeptides, which can.have the same or different primary sequence. In some. cases, the linker used to tether the GH
supergene family member, and/or the linked non-GH supergene family member, polypeptides together can be a bifunctional PEG reagent.
1004931 In some embodiments, the methods and compositions described herein provide for water-soluble bifunctional Gnkers that have a dumbbell structure that includes: a) an azide, an alkyne, a hydrazine, a diamine, a hydrazide, a hydroxylamine, or a carbonyl (including a dicarbonyl)-containing moiety on at least a first end of a polymer backbone; and b) at least a second functional group on a second end of the polymer backbone. 'fhe second functional group can be the same or different as the first functional group. The second functional group, in some embodiments, is not reactive with the first functional group. The methods and compositions described herein provide, in some embodiments, water-soluble compounds that comprise at least one arm of a branched molecular structure. For example, the branched molecular structure can be dendritic.
1004941 In some embodiments, the methods and compositions described herein provide multimers comprising one or niore GI-I supergene family member formed by reactions with water soluble activated polymers that have the structure:
R-(CHaCH2O)n-O-(CHZ)n; X
1004951 wherein n is from about 5 to about 3,000, m is 2-10, X can be an azide, an alkyne, a hydrazine, a diamine, a hydrazide, a hydroxylamine, a acetyl, or carbonyl (including a dicarbonyl)-containing moiety, and R
is a capping group, a functional group, or a leaving group that can be the same or different as X. R can be, for example, a functional group selected from the group consisting of hydroxyl, protected hydroxyl, alkoxyl, N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester, N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate, acrylamid'e, active sulfone, amine, aminooxy, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide; epoxide, glyoxals, diones, mesylates, tosylates, and tresylate, alkene, and ketone. In a further embodiment, linker groups can be used to link transcription factors. Genes require multiple transcription factors to efficiently initiate expression of the encoded protein. Transcription factors synthesized with non-natural aniino acids can be linked, via linkers as described above, and used to enhance artificial activation of targeted genes. The linked transcription factors can bind target DNA and promote recruitment of RNA polymerase in the absence of the nornial activation signal cascade, thus expressing genes without the required signal. In yet a further embodiment, ligands for cell receptors can be linked for efficient activation of the receptors. Platelet-derived Growth Factor (PDGF) forms dimers in order to bind its receptor. PDGF
which contains non-natural amino acids can be linked in dimer formation via linkers as described above and administered to provide efficient binding of the PDGF receptor. Still further embodiments of linked proteins include linked antibodies.
Two different antibodies, each specific for unique epitopes on the same or adjacent =targets. can be linked for enhanced stimulation, binding, or neutralization. For example, antibodies specific for two different epitopes found on gp120 and associated gp4O of HIV can be linked to provide more:
effectiveneutralization of the target.
Similarly, linked antibodies can be used to stimulate cell surface receptors.
For example, antibodies to CD3 as well as CD4 of the T-cell receptor can be linked to provide the necessary stimuli for activation of the receptor.

A further embodiment includes peptides linked to nucleic acid. For example, ligands for cell receptors or proteins which bind cell surfaces can be linked to a therapeutic nucleic acid that is administered to a desired target. The linked ligand facilitates the uptake of the nucleic acid that is then expressed within the cell to exert its therapeutic effect. Similarly, peptides may be linked to nucleic acids to facilitate packaging or condensation of the nucleic acid.
1004961 The functional groups on the linker do not have to be identical, nor do they have to be phenyl hydrazine groups. The chemistry detailed throughout this specification allows the design of a linker in which at least one functional group can form an indole group with a non-natural amino acid polypeptide; the other functional groups on the linker, in some. embodiments, utilize other documented methodologies, including nucleophile/electrophile based cheniistry.

C. Exanrple ofAdding Functionality: Easing the Isolation Properties of a Polypept[de 1004971 A naturally-occutring or non-natural amino acid polypeptide may be difficult to isolate from a sample for a number of reasons, including but not limited to the solubility or binding characteristics of the polypeptide.
For example, in the preparation of a polypeptide for therapeutic use; such a polypeptide may be isolated from a recombinant system that lias been engineered to overproduce the polypeptide.
However, because of the solubility or binding characteristics of the polypeptide, achieving a desired level of purity often proves difficult.
The methods, compositions, techniques and strategies described herein provide a solution to this situation.
1004981 The methods, compositions, techniques and strategies described herein, allow production of a heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide that is homologous to the desired polypeptide, wherein the heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide has improved isolation characteristics. In one embodiment, a homologous non-natural amino acid polypeptide is produced biosynthetically. In a further or additional embodinient, the non-natural amino acid has incorporated into its structure one of the non-natural amino acids described herein. In a further or additional embodiment, the non-natural aniino acid is incorporated at a terminal or internal position and is further incorporated site specifically.
1004991 In one embodiment, the resulting non-natural amino acid, as produced biosynthetically, already has the desired improved isolation characteristics. In further or additional embodiments, the non-natural amino acid comprises a heterocycle, including a nitrogen-containing heterocycle, linkage to a group that provides the improved isolation characteristics. In further or additional embodiments, the non-natural amino acid is further modified to form a modified heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide, wherein the modification provides a heterocycle, including a nitrogen-containing heterocycle, linkage to a group that provides the improved isolation characteristics. ln some embodiments, such a group is directly linked to the non-natural amino acid, and in other embodiments, such a group is linked via a linker group to the non-natural amino acid. In certain embodiments, such a group is connected to the non-natural amino acid by a single chemical reaction, in other embodiments a series of chemical reactions is required to connect such a group to the non-natural amino acid. Preferably, the group imparting improved isolation characteristics is linked site specifically to the non-natural amino acid in the non-natural amino acid polypeptide and is not linked to a naturally occuring amino acid under the reaction conditions utilized.
1005001 In further or additional embodiments the resulting non-natural amino acid polypeptide is homologous to the GH supergene family members, however, the methods, techniques and composirions described in this section can be applied to virtually any other polypeptide which can benefit from improved isolation characteristics, including by way of example only, a therapeutic protein.
1005011 In further or additional embodiments, the group imparting improved isolation characteristics improves the water solubility of the polypeptide; in other embodiments, the group improves the binding properties of the polypeptide; in other embodiments, the group provides new binding properties to the polypeptide (including, by way of example only, a biotin group or a biotin-binding group). In embodiments wherein the group improves the water solubility of the polypeptide, the group is selected from the water soluble polymers described herein, including by way of exarnple only, any of the PEG polymer groups described herein.

B. Example of Adding Fiuictiojtality: Detecting the Presence of a Polypeptide 1005021 A naturally-occurring or non-natural amino acid polypeptide may be difficult to detect in a sample (including an in vivo sample and an in vitro sample) for a number of reasons, including but not limited to the lack of a reagent or label that can readily bind to the polypeptide. The methods, compositions, techniques and strategies described herein provide a solution to this situation.
[005031 The methods, contpositions, techniques and strategies described herein allow production of a heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide that is homologous to the desired polypeptide, wherein the heterocycle-, including a nitrogen-containing heterocycle, containing non-natural anvno acid polypeptide allows the detection of the polypeptide in an in vivo sample and an in vitro sample. In one embodiment, a homologous non-natural amino acid polypeptide is produced biosynthetically. In a further or additional embodiment, the non-natural amino acid has incorporated into its structure one of the non-natural amino acids described herein. In a further or additioal embodiment, the non-natural amino acid is incorporated at a terminal or internal position and is further incorporated site specifically.
1005041 In one embodiment, the resulting non-natural amino acid polypeptide, as produced biosynthetically, already has the desired detection characteristics. In further or additional embodiments, the non-natural amino acid polypeptide comprises at least one non-natural amino acid selected from the group consisting of a carbonyl-containing non-natural amino acid, a hydrazine-containing non-natural amino acid, including an indole-containing amino acid to provide improved detection characteristics. In other embodiments such non-natural aniino acids have been biosynthetically incorporated into the polypeptide as described herein. In further or alternative enibodiments non-natural amino acid polypeptide comprises at ]east one non-natural ainino acid selected from amino acids of Formula I-XV. In further or alternative embodiments non-natural amino acid polypeptide comprises at least on e non-natural amino acid selected from amino acids of compounds of structures 1-4. In further or additional embodiments, the non-natural amino acid comprises an indole linkage that provides the improved detection characteristics. In further or additional embodiments, the non-natural amino acid is further nlodified to form a modified indole-containing non-natural amino acid polypeptide, wherein the modification provides an indole-containing linkage to a group that provides the improved detection characteristics. In some embodiments, such a group is directly linked to the non-natural amino acid, and in other embodiments, such a group is linked via a linker group to the non-natural aniino acid. In certain einbodiments, such a group is connected to the non-natural aniino acid by a single chemical reaction, in other embodinients a series of chemical reactions is required to connect such a group to the non-natural amino acid. Preferably, the group imparting improved detection characteristics is linked site specifically to the non-natural amino acid in the non-natural amino acid polypeptide and is not linked to a naturally occurring amino acid under the reaction conditions utilized.
1005051 In further or additional embodiments the resulting non-natural amino acid polypeptide is homologous to the GH supergene family members, however, the methods, techniques and compositions described in this section can be applied to virtually any other polypeptide which needs to be detected.in an in vivo sample and an in vitro sample, including.by way of example only, a therapeutic protein.
1005061 In further or additional enibodiments, the group imparting improved detection characteristics is selected froni the group consisting of a label; a dye; an affinity label; a photoaffinity label; a spin label; a fluorophore; a radioactive moiety; a moiety incorporating a heavy atom; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; a chromophore; an energy transfer agent; a detectable label; and any combination thereof.
1005071 In one embodiment, an antibody is engineered to contain an indole non-natural amino acid, and the antibody recognizes a unique antigen on a cancerous cell. After labeling/or modifying the antibody with the indole-via methods described herein and purifying the labelled antibody, it is administered to a subject suspected of having a cancer that can be recognizcd by the labelled antibody.
Following administration of the labelled antibody, presence and location of the labelled antibody within the patient can identify the presence of cancerous tissues. Administration of the labelled antibody allows for the detection of the cancer within the patient, metasteses within the subject, and/or efficacy of treatments for the cancer within the subject.
1005081 In another embodiment, a peptide that binds to antigens on the surface of cells is engineered to contain a dye, inlcuding but not linuted to fluorescent dyes which can be used to track the peptide following administration of the peptide to a subject. The dye is attached to the peptide via the non-natural amino acid located within the peptide, and the peptide is administered to the subject.
Localization or binding of the peptide to its ligand(s) is accomplished with documented imaging or detection techniques.
1005091 In another embodiment is a method for detecting the presence of a polypeptide in a patient, the method comprising administering an effective amount of a homologous non-natttral atnino acid polypeptide comprising at least one non-natural amino acid having the structures of conipounds 1-4:
H
R K3 AR.~\ N R3 R3 A B af\ \ N Ra R 3 R- ~/ ~ R,.N RZ n Rs rH~ 1 RS HRaG R. m~ R
O 2 s Ra N Rs Rs R
R s Ra n\ s N Rs R3 R3 A, R5 R3 A'B'~
B m R Rs s Rl~ R, Ra R, ~N RZ
H~ O 3 H R4 0 4 R5 Rs wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker, linked at one end to an indole-containing nioiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower.heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)z-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or-subs6tuted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNOZ)N(R')-, -N(R')C(NCOOR')N(R')-, -N(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')Z-N=N-, and -C(R'),-N(R')-N(R')-and each R' is independently H, alkyl, or substituted alkyl;
R~ is H, an amino protecting group, resin, at least one amino acid, polvpeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
n is 0, I, 2, or 3, and m is 0, 1, 2, or 3, provided that at least one of n or m is not 0;
wherein, each ring in structures 1, 2, 3, and 4 that has an associated R.
group can contain 0, 1, or 2 Re groups and each R. is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")z, -OR", and -S(O)kR", where.k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl; or when more than one R. group is present, two R. may optionally form an aryl, cycloalkyl or heterocycloalkyl;
each of R3.and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and .R4 or two R, groups optionally form a cycloalkyl or a heterocycloalkyl;
each R5 is independently H, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted.alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide,.aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, OH, NH2, CN; NOZ, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R", -C(0)2R", or -C(O)N(R")Z, wherein each R" is independently liydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
or RS is L-X, where, X is a selected from the group consisting of: a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic contpound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypcptide or polypeptide analog; an antibody or antibody fragment; a nietal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moie.ty; a radioactive moiety; a novel functional group; agroup that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety;
an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating,group; a chromophore;
an energy transfer agent; a biologically active agent; a detectable label; a small molecule; an inhibitory ribomicleic acid; a radionucleotide; a neutron-capture agent; a derivative of biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an abzyme, an activated complex activator,-a vinis, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1,2, or 3, -S(O)k(alkylene orsubstituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-,. -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -(alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)-S(O)k-( alkylene or substituted alkylene)-S-, -(alkylene or substituted alkylene)-S-S-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
when more than one R$ group is present, two ortho R5 groups can optionally form a heterocycloalkyl or an aromatic heterocycloalkyl;
or the -B-indole containing moiety together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable prodrug or solvate thereof.
1005101 In another enibodiment is a method for detecting the presence of a polypeptide in a patient, the method comprising adntinistering an effective amount of a homologous non-natural amino acid polypeptide comprising at least one non-natural amino acid having the structures of compounds 1-4, wherein the at least one non-natural acid is incorporated at a specific site within the polypeptide. In yet another embodiment is a method for detecting the presence of a polypeptide in a patient, the method comprising administering an effective amount of -a homologous non-natural amino acid polypeptide comprising at least one non-natural amino acid having the stnictures of compounds 1-4, whercin the non-natural amino acid is incorporated using a translation system. In another embodiment is a method for detecting the presence of a polypeptide in a patient, the method comprising administering an effective amount of a homologous non-natural amino acid polypeptide comprising at least one non-natural amino acid having the structures of compounds 1-4, wherein the non-natural amino acid is incorporated into the polypeptide using a translation system and a post translation modification system. In another embodiment is a method for detecting the presence of a polypeptide in a patient, the method comprising administering an effective=amount.of a homologous non-natural amino acid polypeptide cotriprising at least one non-natural amino acid having the structures of compounds 1-4, wherein the at least one non-natural amino acid is_stable: in aqueous.solution for at least I month. ln another eniboditnent is a method for detecting the presence of a polypeptide in a patient, the method coniprising administeringan effective amount of a homologous non-natural amino acid polypeptide comprising at least one non-natural amino acid having the.stiuctttres of compounds 1-4, wherein the at least one non-natural amino acid is stable for at least 2 weeks. In another embodiment is a method for detecting the presence ofapolypeptide in a patient, the method-comprising administering an effective amount of a homologous non-natural aniino acid polypeptide comprising at least one non-natural amino acid having the structures of compounds 1-4, wherein the at least one non-natural amino acid is stable for at least 5 days.
1005111 In another embodiment is a method for detecting the presence of a polypeptide in a patient, the method comprising administering an effective amount of a homologous non-natural amino acid polypeptide comprising at least one non-natural amino acid having the structures of compounds 1-4, wherein the polypeptide is a protein homologous to a therapeutic protein selected from the group consisting of:
alpha-I antitrypsin., angiostatin, antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD401igand, c-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidernial growth factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), exfoliating toxin, Factor IXjactor VA, Factor VIII, Factor X, fibroblasrgrowth factor (FGF), fibrinogen, fibronectin, four-helical bundle protein, G-CSF, glp-l, GM-CSF,;glucocerebrosidase,,gonadotropin, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), hurnan serutn albuniin, ICAM-1, ICAM-I receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-1, IGF-I1, interferon (WN), IFN-alpha, IFN-beta, IFN-gamma, interieukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, (L-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukenva inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide homione, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SECI, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.
1005121 In yet another enibodiment, an indole-containing moiety is attached to a peptide, polypeptide, or protein via non-natural amino acids located within the peptide, polypeptide, or protein. Appropriately labelled peptides, polypeptides, or proteins are adniinistered to a desired subject for detection and imaging via documented methodologies. Through these labelled peptides,. polypeptides, or proteins, a variety of diseases, metabolic pathways, physiological structures or cellular components can be imaged. By way of example, fluorescent imaging microscopy can be used to detect the presence of labelled peptides, polypeptides, or proteins within a subject.

C. Example of Adding Functionality: I-nproving the Tlrerapeutic Properties of a Polypeptide 1005131 A naturally-occurring or non-natural amino acid polypeptide will be able to provide a certain therapeutic benefit to a patient with a particular disorder, disease or condition. Such a therapeutic benefit will depend upon a number of factors, including by way of example only: the safety profile.of the polypeptide, and the pharmacokinetics, pharmacologics and/or pharmacodynamics of the polypeptide (e.g., water solubility, bioavailability, serum half-life, therapeutic half-life, immunogenicity, biological activity, or circulation time). In addition, it may be advantageous to provide additional functionality to the polypeptide, such as an attached cytotoxic compound or drug, or it may be desirable to attach additional polypeptides to form the homo- and heteromultimers described herein. Such niodifications preferably do not destroy the activity and/or tertiary structure of the original polypeptide. The methods, compositions, techniques and strategies described herein provide solutions to these issues.
1005141 The methods, compositions, techniques and strategies described allow production of a heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide that is homologous to the desired polypeptide, wherein the heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide has improved therapeutic characteristics.
In one embodiment, a honiologous non-natural amino acid polypeptide is produced biosynthetically. In a further or additional embodiment, the non-natural amino acid has incorporated into its structure one of the non-natural amino acids described herein.
In a further or additional embodiment, the non-natural anrino acid is incorporated at a terminal or internal position and is further incorporated site specifically.
1005151 In one embodiment, the resulting non-natural amino acid, as produced biosynthetically, already has the desired improved therapeutic characteristics. In further or additional embodiments, the non-natural amino acid comprises a heterocycle, including a nitrogen-containing heterocycle, linkage to a group that provides the improved therapeutic characteristics. In further or additional embodiments, the non-natural amino acid is further niodified to form a modified heterocycle-, including a nitrogen-containing heterocycle, containing non-natural atnino acid polypeptide, wherein the modification provides a heterocycle, including a nitrogen-containing heterocycle, linkage to a group that provides the iniproved therapeutic characteristics. In some embodiments, such a group is directly linked to the non-natural amino acid, and in other embodiments, such a group is liiilced via a linker group to the non-natural amino acid. In certain embodiments, sucli a group is connected to the non-natural amino acid by a single chemical reaction, in other embodintents a series of chcniical reactions is required to connect such a group to the non-natural amino acid. Preferably, the group imparting improved therapeutic characteristics is linked site specifically to the non-natural amino acid in the non-natural amino acid polypeptide and is not linked to a naturally occurring amino acid under the reaction conditions utilized.
1005161 In further or additional embodiments the resulting non-natural amino acid polypeptide is homologous to the GH supergene family members, however, the methods, techniques and compositions described in this section can be applied to virtually any other polypeptide which can benefit from improved therapeutic characteristics, including by way of example only, a therapeutic protein.
1005171 In further or additional embodiments, the group imparting improved therapeutic characteristics improves the water solubility of the polypeptide; in other embodiments, the group improves the binding properties of the polypeptide; in other embodiments, the group provides new binding properties to the polypeptide (including, by way of example only, a biotin group or a biotin-binding group). In embodiments wherein the group improves the water solubility of the polypeptide, the group is selected from the water soluble polyniers described herein, including by way of example only the PEG polymer groups. In further or additional embodiments the group is a cytotoxic compound, whereas in other embodiments the group is a drug. In further emboditnents the linked drug or cytotoxic compound can be cleaved from the non-natural amino acid polypeptide so as to deliver the drug or cytotoxic compound to a desired therapeutic location. In othe embodiments, the group is a second polypeptide, including by way of example, a heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide, further including by way of example, a polypeptide that has the same amino acid structure as the first non-natural amino acid polypeptide.
[005181 In further or additional embodiments, the heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide is a modified heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide. In further or additional embodiments, the heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide increases the bioavailability of the polypeptide relative to the liomologous naturally-occumng amino acid polypeptide. In further or additional embodiments, the heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide increases the safety profile of the polypeptide relative to the homologous naturally-occurring atnino acid polypeptide. In further or additional embodinients, the heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide increases the water solubility of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide. In further or additional embodiments, the heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide increases the therapeutic half-life of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide. In further or additional embodiments, the heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide increases the serum half-life of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide. In further or additional embodiments, the heterocycle-, including a nitrogen-containing heterocycle, containing non-nahiral amino acid polypeptide extends the circulation time of the polypeptide relative to the. homologous naturally-occurring amino acid polypeptide. In further or additional embodiments, the heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide modulates the biological activity=of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide. In further or additional embodiments, the heterocycle-, including a nitrogen-containing heterocycle, containing non-natural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide.

XI. Therapeutic Uses of Modified Polypeptides 1005191 For convenience, the modified or unmodified non-natural. amino acid polypeptides described in this section have been described generically and/or with specific examples.
However, the modified or unmodified non-natural amino acid polypeptides described in this section should not. be limited to just the generic descriptions or specific example provided in this section, but rather the modified or unmodified non-natural amino acid polypeptides described in this section apply equally well to all modified or unmodified non-natural amino acid polypeptides comprising at least one non-natural amino acid which falls within the scope of Formulas I-XV and compounds of structures 1-4, including any sub-formulas or specific compounds that fall within the scope of Formulas I-LXV, that are described:in the specification, claims and figures herein.
1005201 The modified or unmodified non-natural amino acid polypeptides described herein, including homo-and hetero-multimers thereof find tnu7tiple uses, including but not limited.
to: therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy-production, consumer products, and/or military uses. As a non-liiniting illustration, the following therapeutic uses of modified or unmodified non-natural amino acid polypeptides are provided.
1005211 The modified or unmodified non-natural amino acid polypeptides described herein are useful for treating a wide range of disorders, conditions or diseases. Administration of the modified or unmodified non-natural amino acid polypeptide products described herein results in any of the activities demonstrated by comnlercially available polypeptide preparations in humans. Average qttantities of the modified or unmodified non-natural amino acid polypeptide product nzay vary and in particular should be based upon the recommendations and prescription of a qualified physician. The exact amount of the modified or unmodified non-natural amino acid polypeptide is a matter of preference subject to such factors as the exact type of condition being treated, the condition of the patient being treated, as well as the other ingredients in the composition. The amount to be given may be determined based upon therapy with the modified or unmodified non-natural amino acid polypeptide.

A. rldarinistration and Pharneaceutical Compositions 1005221 The non-natural amino acid polypeptides, modified or unmodified, as described lierein (including but not liniited to, synthetases, proteins comprising one or more non-natural anuno acid, etc.) are optionally employed for therapeutic uses, including but not lirnited to, in combination with a suitable pharmaceutical carrier. Such compositions, for example, comprise a therapeutically effective amount of the non-natural amino acid polypeptides, modified or unmodified, as described herein, and a pharmaceutically acceptable carrier or excipient. Such a carrier or excipient includes, but is not limited to;
saline, buffered saline, dextrose, water, glycerol, ethanol, and/or combinations thereof. The fornlulation is tnade to suit the mode of administration. In general, methods of adnunistering proteins can be applied to administration of the non-natural amino acid polypeptides, modified or unmodified, as described herein.
1005231 Therapeutic compositions comprising one or more of the non-natural amino acid polypeptides, tnodified or unniodified, as described herein are optionally tested in one or more appropriate in vitro and/or in vivo animal models of disease, to confirm efficacy, tissue metabolism, and to estimate dosages, according to documented methodologies. In particular, dosages can be initially determined by activity, stability or other suitable measures of non-natural to natural amino acid homologues (including but not limited to, cotnparison of a polypeptide modified to include one or more non-natural amino acids to a natural amino acid polypeptide), i.e., in a relevant assay.
1005241 Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. The non-natural amino acid polypeptides, modified or unmodified, as described herein, are administered in any suitable manner, optionally with one or more pharniaceutically acceptable carriers. Suitable methods of administering the non-natural amino acid polypeptides, modified or unmodified, as described herein, to a patient are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective action or reaction than another route.
1005251 Pharmaceutically acceptable carriers are detemiined in part by the particular composition being administered, as well as by the particular niethod used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions described herein.
1005261 The non-natural anuno acid polypeptides described herein and compositions comprising such polypeptides may be administered by any route suitable for proteins or peptides, including, but not limited to parenterally, e:g: injections including, but not limited to, subcutaneously or intraveriously or any other form of injections or.infusions. Polypeptide pharmaceutical compositions (including the various non-riatural amino acid polypeptides described herein) can be administered by a number of routes including, but not limited.,to oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means.
Compositions comprising non-natural amino.acid polypeptides, modified or unmodified, as described herein, can also be administered via liposomes. The non-natural amino acid polypeptides described herein may be used alone or in combination with other suitable components, including but not limited to, a pharmaceutical carrier.
1005271 The non-natural amino acid polypeptides, modified or unmodified, as described herein, alone or in combination with other suitable components, can also be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
1005281 Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the. intended recipient, and aqueous and non-aqueous sterile suspensions thatcan include-suspending agents,,solubilizers, thickening agents, stabilizers, and preservatives. The formulations of packaged nucleic acid can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
1005291 Parenteral administration and'intravenous administration are.
preferred methods of adnunistration. In particular, the routes of administration already in use for natural anuno acid homologue therapeutics (including but not limited to, those typically used for EPO, IFN, GH, G-CSF, GM-CSF, IFNs, interleukins, antibodies, and/or any other pharmaceutically delivered protein), along with formulations in current use, provide preferred routes of administration and formulation for the non-natural amino acid polypeptides, modified or unmodified, as described herein.

1005301 The. dose adtninistered to a patient, in the context compositions and.
methods described herein, is sufficient to have a beneficial therapeutic response in the, patient over time. The dose is deternuned by the efficacy of the particular formulation, and the activity, stability or serum half-life of the non-natural amino acid polypeptides, modified or unmodified, employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose is also determined by the existence, nature, and extent of any adverse side-effects-that accompany the administration of a particular formulation, or the like in a particular patient.
1005311 Tn determining the effective amount of the formulation to be administered in the treatment or prophylaxis.of disease (including but not limited to, cancers, inherited diseases, diabetes, AIDS, or the like), the physician evaluates circulating plasnia levels, formulation toxicities, progression of the disease, and/or where relevant, the production of anti-non-natural amino acid polypeptide antibodies.
1005321 The dose adniinistered, for example, to a 70 kilogram patient, is typically in the range equivalent to dosages of currently-used therapeutic proteins, adjusted for the altered activity or serum half-life of the relevant composition. The pharmaceutical formulations described herein can supplement a variety of therapies, including antibody administration, vaccine administration, administration of cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogues, biologic response modifiers, and the like.
1005331 For adnunistration, the pharmaceutical fotYrtulations described herein are administered at a rate determined by the LD-50 or ED-50 of the relevant formulation, and/or observation of any side-effects of the non-natural amino acid polypeptides, modified or unmodified, at various concentrations, including but not limited to, as applied to the mass and overall health of the patient.
Administration can be. accomplished via single or divided doses.
1005341 If a patient undergoing infusion of a formulation develops fevers, chills, or muscle aches, he/she receives the appropriate dose of aspirin, ibuprofen, acetaminophen or other pain/fever controlling drug. Patients who experience reactions to the infusion such as fever, muscle aches, and chills are. premedicated 30 minutes prior to the future infusions with either aspirin, acetatninophen, or, including but not limited to, diphenhydramine. Meperidine is used for more severe chills and muscle aches that do not quickly respond to antipyretics and antihistamines. Cell infusion is slowed or discontinued depending upon the severity of the reaction.
1005351 Non-natural amino acid polypeptides, modified or unmodified, as described herein, can be administered directly to a mammalian subject. Administration is by any of the routes normally used for introducing a polypeptide to a subject. The non-natural amino acid polypeptides, modified or unmodified, as described herein, include those suitable for oral, rectal, topical, inhalation (including but not limited to, via an aerosol), buccal (including. but not limited to, sub-lingual), vaginal, parenteral (including but not limited to, subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, inracerebral, intraarterial, or intravenous), topical (i.e_ both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the.most suitable route in any given case will depend on the nature and severity of the condition being treated. Administration can be either local or' systemic. T7le formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. The non-natural amino acid polypeptides, modified or unmodified, as described herein, can be prepared in a mixture in a unit dosage injectable form (including but not limited to, solution, suspension, or emulsion) with a pharmaceutically acceptable carrier. The non-natural amino acid polypeptides, modified or unmodified, as described herein, can also be administered by continuous infusion (using, including but not limited to, minipumps such as osmotic pumps), single bolus or slow-release depot.formulations.
1005361 Formulatioils suitable for administration include aqueous and:non-aqueous solutions, isotonicsterile solutions, which can;contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Solutions and.suspensions can be.prepared from sterile:powders, granules, and tablets of the kind previously described.
1005371 Freeze-drying is a commonly employed technique for presenting proteins which serves to remove water from the, protein preparation of interest. Freeze-drying, or lyophilization, is a process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environmerit. An excipient may be included in pre-lyophilized fornwlations to enhance stability during the freeze-drying process and/or to improve stability of the lyophilizcd product upon storage. Pikal, M. Biopharm.
3(9) 26-30 (1990) and Arakawa et al. Pharm. Res. 8(3):285-291 (1991).
1005381 The spray drying of phannaceuticals is documented. For example, see Broadhead, J. et al., "The Spray Drying of Pharmaceuticals," in Drug Dev. Ind. Pharm, 18 (11 & 12), 1169-1206 (1992). In addition to small molecule pharmaceuticals, a variety of biological materials have been spray dried and these include: enzymes, sera, plasma, micro-organisms and yeasts. Spray drying is a useful technique because it can convert a liquid pharmaceutical preparation into a fine, dustless or agglomerated powder 'in a one-step process. The basic technique comprises the following four.steps: a) atomization of the feed solution into a spray; b) spray-air 20. contact; c) drying of the spray; and d) separation of the dried product from the drying air. U:S.. Patent Nos.
6,235,710 and 6,001,800, which. are herein incorporated by reference for the aforementioned disclosure, describe the: preparation of recombinant erythropoietin by spray drying.
1005391 The pkiarmaceutical compositions described herein may 'comprise apharmaceutically acceptable carrier, excipient or stabilizer. Pharmaceutically acceptable carriers are deternuned in part by the particular composition being administered, as well as by the particular method used to administer the composition.
Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions (including optional pharmaceutically acceptable carriers, excipients, or stabilizers) for the non-natural amino acid polypeptides, niodiGed or unmodified, described herein, (see, for example, in Remington: The Science and Practice of Phannacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical.Sciences, Mack Publishing Co., Easton, Pennsylvania 19.75;
Liberman, H.A. and Lachinan, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pliarmaceutical Dosage Forms and Drug Delivery Systenis, Seventh Ed. (Lippincott Willianis & Wilkins, 1999)). Suitable carriers include buffers containing succinate, phosphate, borate, HEPES, citrate, imidazole, acetate; bicarbonate, and other organic acids; antioxidants including but not limited to, ascorbic acid; low molecular weight polypeptides including but not limited to those less than about 10 residues; proteins, including but not limited to, serum.
albumin, getatin, or immunoglobulins; hydrophilic polymers including but not limited to, polyvinylpyrrolidone;
amino acids including but not linvted to, glycine, glutamine,. asparagine, arginine, histidine or histidine derivatives, methionine, glutamate, or lysine; monosaccharides, disaccharides, and other carbohydrates, including but not limited to, trehalose, sucrose, glucose, mannose, or dextrins; chelating agents including but not limited to, EDTA and edentate disodium; divalent metal ions including but not limited to,, zinc, cobalt; or copper; sugar alcohols including but not limited to, mannitol or sorbitol;
salt-forming'counter ions including but not limited to, sodium; and/or nonionic surfactants, including but not limited to TweenTM (including but not limited to, Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20), PluronicsT"' and other pluronic acids, including but not limited to, and other pluronic acids, including but not limited to, pluronic acid F68 (poloxamer 188), or PEG. Suitable surfactants include for example but are not limited to polyethers based upon poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), i.e., (PEO-PPO-PEO), or poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide), i.e., (PPO-PEO-PPO), or a combination thereof. PEO-PPO-PEO and PPO-PEO-PPO are commercially available under the trade names PluronicsTM, R-PluronicsTM, TetronicsTM and R-TetronicsTM (BASF Wyandotte Corp., Wyandotte, Mich.) and are further described in U.S.
Pat. No. 4,820,352 incorporated herein in its entirety by reference. Other ethylene/polypropylene block polymers may be suitable surfactants. A surfactant or a combination of suifactants may be used to stabilize PEGylated non-natural amino acid polypeptides against.one or more stresses including but not limited to stress that results frotn agitation. Some of the above may be referred to as "bulking agents." Some may also be referred to as "tonicity modifiers." Antimicrobial preservatives may also .be applied for product stability and antimicrobial effectiveness; suitable preservatives include but are not liniited to, benzyl alcohol, benzalkonium chloride, metacresol, methyl/propyl parabene, cresol, and phenol, or a combination thereof.
1005401 The non-natural amino acid polypeptides, modified or unmodified, as described herein, including those linked to water soluble polymers such as PEG can also be administered by or as part of sustained-release systems. Sustained-release compositions include, including but not limited to, semi-permeable polymer inatrices in the form of shaped articles, including but not limited to, films, or microcapsules. Sustained-release matrices include from biocompatible materials such as poly(2-hydroxyethyl methacrylate) (Langer et al., J. Bianed.
Mater. Res., 15: 267-277 (1981); Langer, Clieui. Tech., 12: 98-105 (1982), ethylene vinyl acetate (Langer et al., supra) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988), polylactides (polylactic acid) (U.S. Patent No.
3,7731919; EP 58,481), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid) polyanhydrides, copolymers of L-glutamic acid and gamma-ethyl=L-glutamate (U.
Sidman et al.., Biopolymers, 22, 547-556 (1983), poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalaiiine, tyrosine,. isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. Sustained-release compositions also include a liposomally entrapped compound. Liposomes containing the compound are prepared by methods such as:
DE 3,218,121; Eppstein et a1., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc.
Natl. Acad. Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 143,949; EP 142,641; Japanese Pat.
Appln. 83-1 1 8008; U.S. Pat. Nos.
4,485,045, 4,619,794, and 4,544,545; and EP 102,324.
1005411 Liposonially entrapped polypeptides can be prepared by methods described in, e.g., DE 3,218,121;
Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; Japanese Pat. Appln. 83-118008; U.S.
Patent Nos. 4,485,045, 5,021,234, and 4,544,545; and EP 102,324. Composition and size of liposomes are documented methodologies. Some examples of liposomes as described in, e.g., Park JW, et al., Proc. Natl.
Acad. Sci. USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D (eds):
MEDICAI. APPLICATIONS OF
LiPosoMEs (1998); Drunimond DC, et al., Liposomal drug delivery systems for cancer therapy, in Teicher B
(ed): CANC6R DRUG DISCOVERY AND DrVELOPMENT (2002); Park JW, et al., Clin.
Cancer Res. 8:1172-1181 (2002); Nielsen UB, et al., Biochim. Biophys: Acta 1591(l or 2):109-118 (2002); Mamot C, et al., Cancer Res.
63:3154-3161 (2003).
The dose administered to a patient in the context of the compositions, formulations and methods described herein, should be sufficient to cause a beneficial response in the subject over time. Generally, the total pharmaceutically effective amount of the non-natural amino acid polypeptides, modified or unmodified, as described herein, administered parenterally per dose is in the range of about 0.01 g/kg/day to about 100 g/kg, or abotit 0.05 mg/kg to about 1 mg/kg, of patient body weight, although this is subject to therapeutic discretion.
The frequency of dosing is also subject to therapeutic discretion, and may be more frequent or less frequent than the commercially available products approved for use in humans. Generally, a polymer:polypeptide conjugate, including by way of example only, a PEGylated polypeptide, as described herein, can be administered by any of the routes of administration described above.
XII. Structure-Function Relationship of Modified Polypeptides 1005421 The non-natural amino acid polypeptides, modified or unmodified, as described herein (including but not limited to, synthetases, proteins comprising one or more non-natural amino acids, etc.) will confer different physical and chemical characteristics on the polypeptide in which it resides.
The usefulness of such characteristics will depend upon the structure of the inon-natural anuno acid, the structure of the modification on the non-natural amino acid, or both, and can be evaluated via experimental models that assess structure-function relationships of test polypeptides.
1005431 In any given experimental model, a non-natural amino acid is substituted for a natural amino acid in a desired polypeptide or protein. After expression of the non-natural amino acid containing peptide or protein, the protein is derivatized with a library of alternate R groups. These R groups are reacted with the non-natural amino acid contained within the polypeptide or protein. The library of R
groups is chosen by their structural or chemical similarity to the R-group of the replaced amino acid. Following the addition of novel R groups to the non-natural amino acid within the protein, the protein is then screened for function or activity within the appropriate test system. By way of example, phenylalanine is replaced with a non-natural aniino acid within a protein. A library of alternative R. groups with similar characteristics to the R group of phenylalanine are then added to the non-natural amino acid. A single altentative R group is added to a non-natural R groups added include rings, hetero-rings, conjugated rings, or other chemical moieties that confer, but not limited to, similar chemical and structural characteristics. The derivitized protein is then screened for function or functions related to the addition of the newly substituted non-natural amino acid by testing in an appropriate experimental model.
Examples of experimental models include, but are not limited to, based assays, cell free assays, cell-based assays, tissue culture models, and animal models.
1005441 In a further embodiinent, indoles are substituted on the non-natural amino acid for pharmacophore activity in drug discovery or as fluorescent cores useful in detection. To facilitate such addition, indole-based R
groups or R groups suitable for indole synthesis are added to the non-natural amino acid by conducting indole formations in aqueous buffers at room temperature with an optomized two-step reaction. Following this reaction, the derivitized'protein is screened for its desired activity.
1005451 By way of example, the effect of non-natural amino acid substitutions in the acid alpha-glucosidase enzyme (GAA) on alleviation of Pompe disease can be evaluated in a mouse model for Pompe disease. A
library of GAA molecules which contain various amino acid substitutions at chosen sites within the enzyme can be created and expressed via the methods and compositions disclosed herein.
The non-natural amino acid containing enzymes can thcn be evaluated for their activity in a mouse model for Pompe disease (mice which are bred to be genetically difficient for GAA (GAA-/-)), either in unmodified or post-translationally modified forms as disclosed herein. The non-natural amino acid containing enzymes can be administered intravenously, orally, or any other route of administration that allows for efficient protein transport and absorption. Efficacy of administration, enzyme half-life, and alleviation of Pompe disease can be evaluated, for example, by measurements of glycogen degredation and/or clearance in the mice, assessment of serum levels of GAA, changes or reduction in cardiomegaly, cardio myopathy, skeletal niyopathy.
1005461 The modified or unmodified non-naturalamino acid polypeptides described herein are useful in a wide range of industiral applications. Use,of the modified or unniodified non-natural amino acid polypeptide:products described herein results in any of the activities demonstrated by conunercially available polypeptide preparations in industrial applications.
1005471 By way of example, enzymes for the production of ethanol can be:
modi6ed with non-natural amino acids and assayed for changes iin function:.A library of alcohol dehydrogenase II and pyruvate decarboxylase enzymes which contain various non-natural amino acid substitutions can be created and expressed via the niethods and compositions disclosed herein. The non-natural aniino acid modified enzymes can then be screened for changes in their efficiency of ethanol production conferred.by or as a result of the non-natural amino acid substitions. Increases in, but not limited to, affinity for substrate and rate of conversion can be screened by documented methodologies, and applied to the industrial production of ethanol.
1005481 Further examples of industrial applieatibn of the. methods and compositions disclosed herein include environmental clean-up of herbicides and pesticides. The removal of a commonly used herbicide, atrazine, from contaminated soil is facilitated by enzymes that metabolize the atrazine, thus rendering it non-toxic. A library of modified antrazine chlorohydrolase enzymes which contain non-natural amino acid substitutions can be created and expressed via the methods and compositions disclosed herein. The library of non-natural amino acid modified atrazine cholorhydrolase enzymes can then be screened for changes in ability to dechlorinate atrazine found in the environment as well as any new modes of atrazine metabolism conferred by or as a result of the non-natural amino-acid substitutions. As described previously, changes in enzyme efficiency can be assessed via documented methodologies; including but not liniited to, increases in metabolism of atrazine or intermediates.

EXAMPLES
H

Example 1 Synthesis of H2N COOH

1005491 The synthesis used is described in the following reaction scheme:
ac ~
~
~ N~ AciO.~. ~ N.N.AC NB3.AIBN I~ N.N.Ac ~/ B k ~ ~ Ac CG< & Ac A,~,~ (COZEI EIONa; EtOH
" -' ~COiEt 3596 for 2 steps ~.{ AC
N-NHZ N;N:Ac FCI. diamne ~ i Ac 81%
Cp:Et HpN COpH AcHN ~Et AC
N.NAc ~
a) Synthesis of 1005501 To a solution of 1-p-tolylhydrazine (5.0 g, 31 mmol) irrpyridine (50 mL) at 0 C was added Ac20 (30 mL, 318 mmol)). The nuxture was stirred at room temperature overnight and quenched with MeOH (100 mL).
After the solvent was renioved in vacuo, the residue was purified by flash chromatography (silica, 20-50%
EtOAc/hexanes) to afford a colorless oil (6.72. g, 87%): 'H NMR (500 MHz, CDCI,) 8 7.28 (d, J= 8.4 Hz, 2H), 7.24 (d, J= 8.4 Hz, 2H), 2.47 (s, 6H), 2.40 (s, 3H), 2.14 (s, 3H); 13C NMR
(125 MHz, CDCI3) S 171.8, 169.5, 139.1, 138.8, 130.4, 126.4, 25.4, 22.3, 21.3.
Ac N,N.Ac b) Synthesis of Br ~Z
[005511 To a solution of N',N'-diacetyl-N-p-tolylacetohydrazide (6.4 g, 25.8 mmol) in CCh (300 mL) was added N-bromo succinimide (5.1 g, 28.7 mmol). The mixture was heated at reflux. 2,2'-Azobisisobutyronitrile (AIBN, 0.2 g, 1.2 mmol) was added. The resultant mixture was stirred at reflux for 36 h and cooled to room temperature. The mixtuie was washed with H20 and brine, dried over anhydrous Na2SO4i filtered and concentrated to afford bromide (8.62 g) as a brown oil. The crude product was directly used for the next step without purification.
Ac F N.N.Ac Ac c) Synthesis of ACHN co2Et 1005521 To a solution of'EtONa (2.3 g, 32.1 nimol) in EtOH (80 mL) was added diethyl 2-acetamidomalonate (6.3 g, 29.0 mmol). The resultant mixture was stirred at. 0 C for 20 min.
N',N'-diacetyl-N-(4-(bromomethyl)phenyl)acetohydrazide (8.62 g, 26.4 nunol) was added in one potion. The mixture was heated at 80 C overnight and cooled to room temperature. Citric acid (10 g, 50 mmol) was added to the reaction mixture.
After most solvent was removed, the residue was diluted with EtOAc (500 mL).
The mixture was washed with H20 and brine, dried over anhydrous NaZSO4, filtered and concentrated. The residue was purified by flash chromatography (silica, 15-80% EtOAc/hexanes) to afford diethyl 2-(4-(ace(amido)benzyl)-2-acetamidotnalonate (4.17 g, 35% for two steps) as a yellow oil: 11=1 NMR (500 MHz, CDC13) S 7.23 (d, J = 8.0 Hz, 2H), 7.03 (d, J = 8.0 Hz, 2H), 6.57 (s, I H.), 4.29-4.20 (m, 4H), 3.65 (m, 2H), 2.41 (s, 6H), 2.08 (s, 3H), 2.01 (s, 3H), 1.27 (t, J= 3.6 Hz, 6H); "C NMR (125 MHz, CDC13) 5 171.7, 169.3, 169:2, 167.4,,140.3, 136.4, 131.3, 126.2, 67.2, 63.0, 37.4, 25.3,,23.2, 22.3, 14.2.
H

d) Synthesis of H2N COOH
1005531 To a solution of diethyl 2-(4-(acetaniido)benzyl)-2-acetamidomalonate (572 mg, 1.24 mmol) in dioxane (15 mL) was added HCI (12 N, 15 mL). The resultant mixture was heated at reflux overnight and concentrated in vacuo. To the residue was added MeOH (1 ml:.). Ether (200 mL) was added to precipitate the product (231 mg, 81%) as a solid: 'H NMR (500 MHz, D20) 8 7.28 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 4.21 (dd, J = 7.4, 5.7 Hz, IH), 3.26 (dd, J = 9.2, 5.7 Hz, 1 H), 3.15 (dd,-J = 14.7, 7.4 Hz, 1H); 13C NMR
(125 MHz, D20) 5 171.5, 142.9, 130.3, 129.0, 115.7, 54.1, 34.7.
Example 2 1005541 The synthesis of this carbonyl-containing amino acid follows standard methodology.
"lO

Example 3 1005551 The synthesis of this carbonyl-containing amino acid follows standard methodology.

O

Example 4: Reaction between an aryl hydrazine and an aldehyde 1005561 Presented in Figures 3, 4, 5, 6, and 7 is a niodel reaction between two molecules to form an indole.
Either of the two starting materials can be present as a sidechain on a non-natural amino acid polypeptide provided that the other reagent represents a functional group on a compound for derivatizing the aforementioned sidechain. That is, the sidechain on a non-natural amino acid polypeptide can be an aryl hydrazine group, and the reactive moiety is an aldehyde connected to a water-soluble polymer, at least one amino acid or a detectable label. Conversely, the sidecliain on a non-natural amino acid polypeptide can be an aldehyde group, and the reactive moiety is an aryl hydrazine connected to a water-soluble polymer, at least one amino acid or a detectable label.

pH 3.6 _ + NNH2 30 H H
4`~

Exaniple 5: Reaction between an aryl hydrazine and an ketone 1005571 Presented in Figures 8, 9, 10, 11, 13 and 14 is a model reaction between two molecules to form an indole. Eitiier of the two starting materials can be present as a sidechain on a non-natural amino acid polypeptide provided that the other reagent represents a functional group on a compound for derivatizing the aforementioned. sidechain. That is, the sidechain on a non-natural antino acid polypeptide can be an aryl hydrazine group, and the reactive moiety is a ketone connected to a water-soluble polymer, at least one amino acid or a. detectable label. Conversely, tlie, sidechain on a non=natural:
amino acid polypeptide can be a ketone group, and the reactive moiety is an aryl hydrazine connected to a water-soluble polymer, at least one amino acid or a detectable label.
Example 6: Urotensin Analogs Containing an Aryl Hydrazine Non-Natural Amino Acid 1005581 Presented in Figure 20 is a Urotensin analog that includes, at the "X"
position, a non-natural amino acid with an aryl hydrazine sidechain. The compound was chemically synthesized using standard techniques. It is to be understood that the sidechain alternatively can include a ketone, an aldehyde, or a protected carbonyl sidecliain (show-n schematically in Figure 21).
Example 7: Indole Formation for the Derivatization of Non-Natural Amino Acids 1005591 Presentcd in Figures 20, 21, 22, 24, 28 and 29 are examples of how the formation of indole moieitics from the reaction of aryl hydrazines with carbonyl groups can be used to derivatize non-natural amino acid polypeptides. The protein `cartoon' shown in these figures can be urotensin, human growth hormone, insulin, an antibody, a kinase, erythropoietin, or any other polypeptide or protein described herein or available in the literature.
Example 8 1005601 This example details cloning and expression of a modified polypeptide in E. coli. An introduced translation system that comprises an brtliogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS) is used to express the polypeptide containing anon-natural amino acid.
The O-RS. preferentially aminoacylates the O-tR1NA with a non-natural amino acid. In turtrn the translation system inserts the non-natural amino acid into the polypeptide, in response to an encoded selector codon.
Amino acid and polynucleotide sequences of.O-tRNA and O-RS useful for'the incorporation of non-natural amino acids are described in U.S.
Patent application serial no. 10/126,927 entitled "In Vivo Incorporation of Unnatural Amino Acids" and U.S.
Patent application serial no. 10/126,931 entitled "Methods and Compositions for the Production of Orthogonal tRNA-Aniinoacyl tRNA Synthetase Pairs," which are incorporated by reference herein.
SEQ ID NO:1 pq, janna.schii n1tRNA~uA tRNA
SEQ ID NO:2 HLAD03; an o timized antber suppressor tRNA tRNA
SEQ ID NO:3 HL325A; ait o timized AGGA ranreshi t su ressor tRNA tRNA
SEQ ID NO:4 Aminoacyl tRNA syn[hetase for tlie incorporation of p-azido-L-phenylalanine RS
Az-PheRS(6) SEQ ID NO:5 Aminoaevl tRNA synthetase for the incorporatiaz of p-benzoyl-L
phenylalanine RS
B aRS l SEQ ID NO:6 Aminoacyl IRNA synt/ietase_/'or the incorporation of propargyl-phenylalanine RS
Prv ar l-PheRS
SEQ ID NO:7 Anzinoacyl tRNA synthetase for the incorporation of propargyl-pheny/alanine RS

Pro ar 1-Phe.RS
SEQ ID NO:8 Aminoacyl tRNA synthetase jor the incotporcitinn ojpropargyl,-phenylalanine RS
Pro ar 1-PheRS
SEQ ID NO:9 Aminoacyl tRNA synthetase for the incorporation of p-azidophenylalanine RS
Az-PheRS I
SEQ ID NO: 10 Aminoacyl tRNA synthetase for the incorporation of p-azido phenylalanine RS
p-Az-PheRS(3) SEQ ID NO:11 Aminoacyl tRNA synthetase for the incorporation of p-azido phenylalcmine RS
Az-PheRS(4 SEQ ID NO: 12 Atninoacy! IRNA synthetase for the incorporation of p-azido phenylalanine RS
-Az-PheRS(2) SEQ ID NO: 13 Aminoacyl tRNA synthetase for the incorporation of p-azido phenylalanine RS
LWI
SEQ ID NO: 14 Aminoacvl tRNA synthetase for the incorporation of p-azido phenylalanine RS
LWS
SEQ ID NO: 15 Arninoacyl tRNA synthetase for the incorporation of p-aziclo-phenylalanine RS
(LW6 SEQ ID NO:16 Aminoacyl tRNA synthetase for tlte incorporation of p-azido phenylalanine RS
(AzPheRS-S
SEQ ID NO'17 Aniinoacyl tRNA synthe.tase for the incorporation of p-azido phenylalanitte RS
(AzPheRS-6) 1005611 The transfomiation of E. coli with plasmids containing the modified gene and the orthogonal aminoacyl tRNA synthetase/tRNA pair (specific for the desired non-natural amino acid) allows the site-specific incorporation of non-natural amino acid into the polypeptide. The transformed E. coli, grown at 37 C in media containing between 0.01 - 100 mM of the particular non-natural amino acid, expresses modified polypeptide with high fidelity and efficiency. The His-tagged polypeptide containing a non-natural amino acid is produced by the E. coli host cells as inclusion bodies or aggregates. The aggregates are solubilized and affinity purified under denaturing conditions in 6M guanidine HCI. Refolding is performed by dialysis at 4`C ovemight in 50mM
TRIS-HCI, pH8.0, 40 M CuSO4, and 2% (w/v) Sarkosyl. The material is then dialyzed against 20mM TRIS-HCI, pH 8.0, 100mM NaC1, 2mM CaC12, followed by removal of the His-tag. See Boissel et al., (1993) 268:15983-93. Methods for purification of polypeptides are are confirmed by SDS-PAGE, Western Blot analyses, or electrospray-ionization ion trap mass spectrometry and the like.
1005621 The following examples describe methods to measure and compare the in vitro and in vivo activity of a modified therapeutically active non-natural amino acid polypeptide to the in vitro and in vivo activity of a therapeutically active natural amino acid polypept9de.
Example 9: Cell Binding Assays 1005631 Cells (3x106) are incubated in duplicate in PBS/1% BSA (100 l) in the absence or presence of various concentrations (volume: 10 l) of unlabeled GH, hGH or GM-CSF and in the presence of 125 I-GH (approx.
100,000 cpm or I ng) at 0 C for 90 minutes (total volume: 120 l). Cells are then resuspended and layered over 200 l ice cold FCS in a 350 lcl plastic centrifuge tube and centrifuged (1000 g; I minute). The pellet is collected by cutting off the end of the tube and pellet and supernatant counted separately in a gamma counter (Packard).
[005641 Specific binding (cpm) is deterrrtined as total binding in the absence of a competitor (inean of duplicates) minus binding(cpm) in the presence of 100-fold excess of unlabeled GH. (non-specific binding). The non-specific binding is measured for each of the cell types used. Experiments are run on separate days using the same preparation of 1ZSI-GH and should display internal consistency. 1251-GH
demonstrates binding to the GH
receptor-producing cells. The binding is inhibited in a dose dependent manner by unlabeled natural GH or hGH, but not by GM-CSF or other negative control. The ability of hGH to compete for the binding of natural 125 1-GH, similar to natural GH, suggests that the receptors recognize both forms equally well.
Example 10: In Vivo Studies of hGH PEGylated via a indole linkage 1005651 PEG-hGH, unmodified hGH and buffer solution are administered to niice or rats. The results show superior activity and prolonged half life of the PEGylated hGH described herein compared to urunodified hGH
which is indicated by significantly increased bodyweight.
Example 11: Measurement of the in vivo Half-life of Conjueated and Non-conjuaated hGH and Variants Thereof.
1005661 All animal experimentation is conducted in an AAALAC accredited facility and under protocols approved by the Institutional Aninial Care and Use Committee of St. Louis University. Rats are housed individually in cages in rooms with a 12-hour light/dark cycle. Aninials are provided access to certified Purina rodent chow 5001 and water ad libitum. For hypophysectomized rats, the drinking water additionally contains 5% glucose.
Example 12: Pharmacokinetic studies 1005671 The quality of each PEGylated mutant hGH is evaluated by three assays before entering animal experiments. The purity of the PEG-hGH (PEGylated via an indole linkage) is examined by running a 4-12%
acrylamide NuPAGE Bis-Tris gel with MES SDS ntnning buffer under non-reducing conditions (Invitrogen, Carlsbad, CA). The gels are stained with Coomassie blue. The PEG-hGH band is greater than 95% pure based on densitometry scan. The endotoxin level in each PEG-hGH is tested by a kinetic LAL assay using the KTA'' kit from Charles River Laboratories (Wihnington, MA), and is less than 5 EU
per dose. The biological activity of the PEG-hGH is assessed with a IM-9 pSTAT5 bioassay, and the EC50 value confirmed to be less than 15 nM.
1005681 Pharmacokinetic properties of PEG-modified growth hormone compounds are compared to each otlier and to nonPEGylated growth hormone in niale Sprague-Dawley rats (261-425g) obtained from Charles River I_aboratories. Catheters are surgically installed into the carotid artery for blood collection. Following successful catheter installation, animals are assigned to treatment groups (three to six per group) prior to dosing. Animals are dosed subcutaneously with 1 mg/kg of compound in a dose volume of 0.41-0.55 nil/kg. Blood samples are collected at various time points via the indwelling catheter and into EDTA-coated microfuge tubes. Plasma is collected after centrifugation, and stored at -80 C until analysis. Compound concentrations are measured using antibody sandwich growth hormone ELISA kits froni either BioSource International (Caniarillo, CA) or Diagnostic Systenis Laboratories (Webster, TX). Concentrations are calculated using standards corresponding to the analog that is dosed. Pharmacokinetic parameters are estimated using the modeling program WinNonlin (Pharsight, version 4.1). Noncompart mental analysis with linear-up/log-down trapezoidal integration is used, and concentration data is unifornily weighted.
1005691 Plasma concentrations are obtained at regular intervals following a single subcutaneous dose in rats.
Rats (n=3-6 per group) are given a single bolus dose of 1 mg/kg protein. hGH
wild-type protein (WHO hGH), His-tagged hGH polypeptide (his-hGH), or His-tagged hGH polypeptide comprising non-natural amino acid indole covalently linked to 30 k.Da PEG at each of six different positions are compared to WHO hGH and (his)-hGH. Plasma samples are taken over the regular time intervals and assayed for injected compound as described.
Concentration vs time curves are evaluated by noncompartmental analysis (Pharsight,. version 4.1). Values shown are averages (+/- standard deviation). Cmax: maximum concentration;
terminal ~l/_,: terniinal half-life;
AUCo.>;,,f: area under the concentration-time curve extrapolated to infnity;
MRT: mean residence time; Cl/f:
apparent total, plasma clearance; Vz/f apparent volume of distribution during terminal phase.
Example 13: Pharmacod,+namic studies 1005701 Hypophysectomized niale Sprague-Dawley rats are obtained from Charles River Laboratories.
Pituitaries are surgically removed at 3-4 weeks of age. Animals re, allowed to acclimate for a period of three weeks, during which time bodyweight was monitored. Atumals with a bodyweight gain of 0-8g over a period of seven days before the start of the study are included and randonrized to treatment groups. Rats are administered either a bolus dose or daily dose subcutaneously. Throughout the study rats re daily and sequentially weighed, anesthetized, bled, and dosed (when applicable). Blood is collected from the orbital sinus using a heparinized capillary tube and placed into an EDTA coated microfuge tube. Plasma is isolated by centrifugation and stored at -80 C until analysis. The mean (+/- S.D.) plasma concentrations are plotted versus time intervals.
1005711 The peptide IGF-1 is a meniber of the family of son-atomedins or insulin-like growth factors. IGF-1 mediates many of the growth-promoting effects of growth hormone. IGF-1 concentrations are measured using a conipetitive binding enzyme immunoassay k-it against the provided rat/mouse IGF-1. standards (Diagnosic Systems Laboratories). Hypophysectomized rats. Rats (n= 5-7 per group) are given either a single dose or daily dose subcutaneously. Animals are sequentially weighed, anesthetized, bled, and dosed (when applicable) daily.
Bodyweight results are taken for placebo, treatments, wild'type hGH (hGH), His-tagged hGH ((his)hGH), and hGH polypeptides comprising an indole covalently-linked to 30 kDa PEG at positions 35 and 92.
Example 14: Human Clinical Trial of the Safety and/or Efficacy of PEGvlated hGH (PEGylated via an indole linkage) Comprising a Non-Naturally Encoded Amino Acid.
[005721 The following example of a clinical trial is used to treat childhood and adult growth hormone deficiency, Tumer syndrome, chronic renal failure, Prader-Willi syndrome, children with intrauterine growth retardation, idiopathic short stature, growth failure associated with clironic high dose glucocorticoid use, post-transplant growth failure, X-linked hypophosphatemic rickets, inflammatory bowel disease, Noonan syndrome, bone dysplasia, growth failure associated with Celiac's disease, muscle wasting associated, e.g., with advance acquired imrnunodeficiency syndrome, promote healing of burns, side effects of severe dieting for obese individuals, fybromyalgia, chronic fatigue syndrome, debilities associated with aging, and other uses of human growth hornione.
1005731 Obiective To compare the safety and pharmacokinetics of subcutaneously administered PEGylated recombinant human hGH comprising a non-naturally encoded aniino acid with one or more of the commercially available. hGH products (including, but not limited to HumatropeT"f (Eli Lilly & Co.), NutropinTb (Genentech), NorditropinThl (Novo-Nordisk), GenotropinTM (Pfizer) and Saizen/SerostimTM
(Serono)).
1005741 Patients Eighteen healthy volunteers ranging between 8-40 years of age and weighing betweett 20-90 kg are enrolled in the study (e.g., children for pediatric indications and adults for adult indications). The subjects will have no clinically significant abnormal laboratory values for hematology or serum chemistry, and a negative urine toxicology screen, HIV screen, and hepatitis B surface antigen.
They should not have any evidence of the following: hypertension; a. history. of any primary heniatologic disease; history of significant hepatic, renal, cardiovascular, gastrointestinal, genitourinary, metabolic, neurologic disease; a history of anemia or seizure disorder; a known sensitivity to bacterial or manunalian-derived products, PEG, or huntan serum albumin; habitual and heavy consumer to beverages containing caffeine;
participation in any other clinical trial or had blood transfused or donated within 30 days of study entry; had exposure to hGH within three months of study entry; had an illness within seven days of study entry; and have significant abnormalities on the pre-study physical examination or the clinical laboratory evaluations within 14 days of study entry. All subjects are evaluated for safety. and all blood collections. for pharmacokinetic analysis are collected as scheduled. All studies are performed with institutional ethics conunittee approval and patient consent.
[005751 Study Design This will be a Phase I, single-center, open-label, randomized,.two-period crossover study in healthy nzale volunteers. Eighteen subjects are randomly assigned to one of two treatment sequence groups (nine subjects/group). GH is adtninistered over two separate dosing periods as a bolus scc. injection in.the upper thigh using equivalent doses of the PEGylated hGH comprising a non-naturally encoded aniino acid and the commercially available,product chosen: The dose and frequency of;administration of the commercially available product is as instructed in the package Iabe1. Additiot>al..do'sing, dosing frequency, or other parameter as desired, using the, commercially. available products may be added to the'study by including additional groups of subjects.
Each dosing period is separated by a 14-day washoutperiod. Subjects are confined to the study center at least 12 houis prior to and. 72 hours following dosing for each of the two dosing periods, but not between dosing periods. Additional.groups of subjects may be added if there are to be additional dosing, frequency, or other parameter, to be tested for the. PEGylated hGH as well. Multiple formulations of GH that are approved for human use niay be used in this study. HumatropeT" (Eli Lilly & Co.), NutropinTM (Genentech), NorditropinTM
(Novo-Nordisk), GenotropinTM (Pfizer) and Saizen/SerostimT"' (Serono)) are commercially available Gl-1 products approved for human use. The experimental formulation of hGH is the PEGylated hGH comprising a non-naturally encoded amino acid.
1005761 Blood Samnline Serial blood is drawn by direct vein puncture before and after administration of hGH.
Venous blood samples (5 mL) for determi.nation of serum GH concentrations are obtained at about 30, 20, and 10 minutes prior to dosing (3 baseline samples) and at approximately the following times. after dosing: 30 minutes and at 1, 2, 5, 8,. 12, 15, 18, 24, 30, 36, 48, 60 and 72 hours. Each serum sample is divided into two aliquots. All serum samples are stored' at -20 C. Serum samples are. shipped on dry ice: Fasting clinical laboratory, tests, (heniatology, serum chemistry, and urinalysis) are performed inunediately prior to the initial dose on day 1, the morning of day 4,immediately prior to dosing on,day 16, and the niorning'of day 19.
1005771 Bioanalytical Methods An ELISA kit procedure (Diagnostic Systems Laboratory [DSL], Webster TX), is used for the deterniination of serum GH concentrations.
[005781 Safety Determinations Vital signs, are recorded immediately prior to each dosing (Days I and 16), and at 6, 24õ 48, and 72 hours after each dosing. Safety determinations are based on the incidence and type of adverse events and the changes in clinical laboratory tests from baseline. In addition, changes froni pre-study in vital sign measurements, including blood pressure, and physical examination results are evaluated.
1005791 Data Analysis Post-dose serum concentration values are corrected. for pre-dose baseline Gl=I
concentrations by subtracting from each of the post-dose values the mean baseline GH concentration determined from averaging the GH levels from the three saniples collected at 30, 20, and 10 minutes. before dosing. Pre-dose seruni GI3 conccntrations are not included in the calculation of the mean value if they are below the quantification levcl of the assay. Pharmacokinetic parameters are determined from serum concentration data corrected for baseline GI-I concentrations. Pharmacokinetic parameters are calculated by model independent methods on a Digital Equipment Corporation VAX 8600 computer system using the latest version of the BIOAVL software. The following pharmacokinetics parameters are determined:
peak serum concentration (C,,,,X); time to peak. serart concentration (t,,,,); area under the,concentration-rirtte curve (AUC) from time zero to the last blood sampling time (AUCa.>>) calculated .with the use of the linear trapezoidal rule; and terminal elimination half-life (tin), computed from the elimination rate constant. The elimination rate constant is estimated by linear regression of consecutive data points in the terminal linear region of the log-linear concentration-time plot. The mean, standard deviation (SD), and coefficient of variation (CV) of the pharmacokinetic parameters are calculated for each treatment. The ratio of the parameter means (preserved formulation/non-preserved formulation) is calculated.
[005801 Safety Results The incidence of adverse events is equally distributed across the treatment groups.
There are no clinically significant changes from baseline or pre-study clinical laboratory tests or blood pressures, and no notable changes from pre-study in physical examination results and vital sign measurements.
The safety profiles for the two treatment groups should appear similar.
1005811 Pharmacokinetic Results Mean senim GH concentration-time profiles (uncorrected for baseline GH
lcvels), in all 18 subjects after receiving a single dose of one or more of comniercially available hGH products (including, but not limited to HumatropeTM (Eli Lilly & Co:), NutropinTm (Genentech), NorditropinT"' (Novo-Nordisk), GenotropinTM (Pfizer) and Saizen/SerostimTM (Serono)) are compared to the PEGylated hGH
comprising a non-naturally encoded amino acid at each time point measured. All subjects should have pre-dose baseline GH concentrations within the normal physiologic range.
Pharrnacokinetic parameters are determined from serum data corrected for pre-dose mean baseline GH concentrations and the C. and t,õ,x are determined.
The mean t,,,ax for the clinical comparator(s) chosen (HumatropeTM (Eli Lilly &-Co.), NutropinTM (Genentech), NorditropinTM (Novo-Nordisk), GenotropinTM (Pfizer), Saizen/SerostimTM
(Serono)) is significantly shorter than the tõm,t for the PEGylated hGH comprising the non-naturally encoded amino acid. Terminal half-life values are significantly shorter for the conunercially available hGH products tested compared with the terminal half-life for the PEGylated hGH comprising a non-naturally encoded amino acid.
1005821 Although the present study is conducted in healthy male subjects, similar absorption characteristics and safety profiles would be anticipated in other patient populations; such as male or female patients with cancer or chronic renal failure, pediatric renal failure patients, patients in autologous predeposit programs, or patients scheduled for elective surgery.
1005831 In conclusion, subcutaneously adniinistered single doses of PEGylated hGH comprising non-naturally encoded amino acid will be safe and well tolerated by healthy male subjects.
Based on a comparative incidence of adverse events, clinical laboratory values, vital signs, and physical examination results, the safety profiles of the commercially available forms of hGH and PEGylated hGH "comprising non-naturally encoded amino acid will be equivalent. The PEGylated hGH comprising non-naturally encoded amino acid potentially provides large clinical utility to patients and health care providers.
Example 15: Comparison of water solubility of PEGylated hGH and non-PEGylated hGH
1005841 The=water solubility of hGH wild-type protein (WHO hGH), His-tagged hGH polypeptide (his-hGH), or His-tagged hGH polypeptide comprising non-natural amino acid indole covalently linked to 30 kDa PEG at position 92 are obtained by determining the quantity of the respective polypeptides which can dissolve on 100 L of water. The quantity of PEGylated hGH is larger than the quantities:for WHO hGH and hGH which shows a that PEGylation ofnon-natural amino acid polypeptides increases the water solubility.

Examnle 16: In Vivo Studies of modified therapeutically active non-natural amino acid polypentide 1005851 Prostate cancer tumor xenografts are implanted uito mice which are "then separated into two groups.
One group is treated daily with a modified therapeutically active non=natural amino acid polypeptide and the other group is treated daily with therapeutically active natural amino acid polypeptide. The tumor size is measured daily and the modified therapeutically active non-natural anuno acid polypeptide has improved therapeutic effectiveness compared to the therapeutically active natural amino acid polypeptide as indicated by a decrease in tumor size for the group treated with the modi6ed therapeutically active non-natural amino acid polypeptide.
Example 17: Measurement of non-natural amino acid polVneptide activity and affinity (005861 This example details the measurement of non-natural amino acid polypeptide activity and affinity of non-natural amino acid polypeptides for their receptor, binding partner, or ligand.
1005871 Protein for the non-natural amino acid polypeptide receptor, binding partner, or ligand is expressed and isolated according to documented methodologies. The BiocoreTM system is used to analyze the binding of non-natural amino acid polypeptide to its receptor. Similarly, a binding partner or ligand may be used in this assay.
1005881 Approximately 600-800 RUs of soluble receptor is immobilized on a BiacoreTM CM5 chip, using an amine-coupling procedure, as recommended by the manufacturer. Various concentrations of wild type or modified or unmodified non-natural amino acid polypeptide in HBS-EP buffer (BiacoreT"t, Pharmacia) are injected over the surface at a flow rate of 40 l/min for 4-5 minutes, and dissociation was monitored for 15 minutes post-injection. The surface is regenerated by a 15 second pulse of 4.5M MgCIZ. Only a minimal loss of binding affinity (1-5%) is observed after at least 100 regeneration cycles: A
reference cell with no receptor inunobilized is used to subtract any buffer bulk effects and non-specific binding.
1005891 Kinetic binding data obtained from modified or unmodified non-natural amino acid polypeptide titration experiments is processed with BiaEvaluation 4.1 software (BIACORETM). Equilibrium dissociation constants (Kd) are calculated as ratios of individual rate constants (k n/kaõ).
1005901 Stable Cell Lines are established expressing receptor, binding partner, or ligand for the non-natural amino acid polypeptide. Cells are electroporated with a coristruct that containing the receptor, binding partner, or ligand cDNA. Transfected cells are allowed to recover for 48 hours before cloning. Receptor, binding partner, or ligand expressing transfectants are identified by surface staining with antibody against the receptor and are analyzed on a FACS Array (BD Biosciences, San Diego, CA). Stably transfected cell clones are established upon further rounds of repeated subcloning of desired transfectants. Such cells are used in cell binding assays.
1005911 Cells (3x106) are incubated in duplicate in PBS/1% BSA (100 l) in the absence or presence of various concentrations (volunie: 10 l) of unlabeled natural amino acid polypeptide or a negative control polypeptide and in the presence of 1251-(modified) non-natural amino acid polypeptide (approx. 100,000 cpm or I ng) at 0 C
for 90 minutes (total volume: 120 l). Cells are then resuspended and layered over 200 l ice cold FCS in a 350 l plastic centrifuge tube and centrifuged (1000 g; 1 minute). The pellet is collected by cutting off the end of the tube and pellet and supernatant counted separately in a ganuna counter (Packard).
1005921 Specific binding (cpm) is determined as total binding in the absence of a competitor (mean of duplicates) minus non-specific binding. The non-specific binding is measured for each of the cell types used.
Experiments are run on separate days using the same preparation of 125I-(modified) non-natural amino acid polypeptide and should display internal consistency. '251-(modified) non-natural aniino acid polypeptide demonstrates binding to the receptor, binding protein, or ligand-producing cells. The binding is inhibited in a dose dependent manner by unlabeled natural amino acid polypeptide, but not by a negative control polypeptide.
1005931 It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof are to be included within the spirit and purview of this application and scope of the appended claims.

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Claims

1. A compound having the structures of compounds 1-4:
wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker, linked at one end to an indole-containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNO2)N(R')-, -N(R')C(NCOOR')N(R')-, -N(R')S(O)k N(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-and each R' is independently H, alkyl, or substituted alkyl;
R1 is H, an amino protecting group or at least one amino acid; and R2 is OH, an ester protecting group or at least one amino acid;
n is 0, 1, 2, or 3, and m is 0, 1, 2, or 3, provided that at least one of n or m is not 0;
wherein, each ring in structures 1, 2, 3, and 4 that has an associated R a group can contain 0, 1, or 2 R a groups and each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)k R", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl; or when more than one R a group is present, two R a may optionally form an aryl, cycloalkyl or heterocycloalkyl;

each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
each R5 is independently H, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, OH, NH2, CN, NO2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R", -C(O)2R", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl,substituted aralkyl, or when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
or R5 is L-X, where, X is a selected from the group consisting of: a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least.one amino acid; at least one sugar group; at least one nucleotide;.at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label;
and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-,-CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -(alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)-S(O)k-(alkylene or substituted alkylene)-S-,-(alkylene or substituted alkylene)-S-S-, -S(O)k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
when more than one R5 group is present, two ortho R5 groups can optionally form a heterocycloalkyl or an aromatic heterocycloalkyl;
or the -B-indole containing moiety together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable prodrug or solvate thereof.

2. The compound of claim 1, wherein A is a bond, substituted or unsubstituted lower alkylene, or an unsubsituted or substituted arylene selected from the group consisting of a phenylene, pyridinylene, pyrimidinylene or thiophenylene.

3. The compound of claim 2, wherein A is a bond.

4. The compound of claim 1, wherein B is a bond, lower alkylene, substituted lower alkylene, -O-(alkylene or substituted alkylene)-, -CON(R")-, -NR'-(alkylene or substituted alkylene)- -N(R")CO-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-, or -S(O)2(alkylene or substituted alkylene)-.

5. The compound of claim 5, wherein B is a bond.

6. The compound of claim 1, wherein R, is at least one amino acid.

7. The compound of claim 1, wherein R2 is at least one amino acid.

8. The compound of claim 7, wherein R1 is at least one amino acid.

9. The compound of claim 1, wherein X is at least one amino acid.

10. The compound of claim 1, wherein X is a detectable label selected from the group consisting of a fluorescent, phosphorescent, chemiluminescent, chelating, electron dense, magnetic, intercalating, radioactive, chromophoric, and energy transfer moiety.

11. The compound of claim 1, wherein X is a water soluble polymer.

12. The compound of claim 11, wherein the water soluble polymer polymer comprises polyalkylene oxide or substituted polyalkylene oxide.

13. The compound of claim 11, wherein the water soluble polymer comprises -[(alkylene or substituted alkylene)-O-(hydrogen, alkyl, or substituted alkyl)]x, wherein x is from 20-10,000.

14. The compound of claim 11, wherein the water soluble polymer is m-PEG
having a molecular weight ranging from about 2 to about 40 KDa.

15. A method of making a compound of structures 1. or 2, comprising reacting a compound of Formula (II) with a carbonyl-containing compound, wherein the compound of Formula (II) is, wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker, linked at one end to an indole containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-,-S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(0)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, =N-O-(alkylene or substituted alkylene), -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)k N(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-;,and each R' is independently H, alkyl, or substituted alkyl;
R1 is H, an amino protecting group, or at least one amino acid; and R2 is OH, an ester protecting group, or at least one amino acid;
each R, is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, CN, NO2, -N(R')2, -C(O)R', -C(O)N(R')2, -OR', and -S(O)k R', where k is 1, 2 or 3 and each R' is independently H, alkyl, or substituted alkyl;
R3 and R4 are independently hydrogen or amine protecting.group, including, but not limited to,

16. The method of claim 15, wherein A is a bond.

17. The method of claim 16, wherein B is a bond.

18. The method of claim 15, wherein R1 is at least one amino acid.

19. The method of claim 15, wherein R2 is at least one amino acid.

20. The method of claim 15, wherein the compound of Formula (II) has the structure of Formula (IV):
wherein, each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, CN, NO2, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)k R", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl.

21. A method of making a compound of structure 3 or 4 comprising reacting a compound of Formula (V) with a hydrazine containing agent; wherein the compound of Formula (V) is:

wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, lower heteroalkenylene, substituted lower heteroalkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k(alkylene or substituted alkylene)-, where k is 1, 2, or 3, -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-;
N(R')CON(R')-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, and -N(R')CO-(alkylene or substituted alkylene)-, where each R' is independently H, alkyl, or substituted alkyl;

J is R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
each R" is independently H, alkyl, substituted alkyl, or a protecting group, or when more.than one R" group is present, two R" optionally form a heterocycloalkyl;
R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and k, is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a carbonyl group, protected carbonyl group, including a protected carbonyl group, or masked carbonyl group, including a masked carbonyl group;
or the -J-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a carbonyl group, protected carbonyl group, including a protected carbonyl group, or masked carbonyl group, including a masked carbonyl group.

22. The method of claim 21, corresponding to Formula (VI):

23. The method of claim 21, corresponding to Formula (VII):
wherein, each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)k R", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl.

24. The method of claim 21, corresponding to Formula (X):
wherein, each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)k R", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl;
Y and Z are independently selected from the group consisting of -OH, alkyl substituted oxygen, -SH or alkyl substituted sulfur and when Y and Z taken together can form a heterocycloalkyl ring.

25. The method of claim 21, corresponding to Formula (XI):
wherein, each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2; -C(O)R", -C(O)N(R")2, -OR", and -S(O)k R", where k is 1, 2, 6r 3, where each R" is independently H, alkyl, or substituted alkyl;
Y is independently selected from the group consisting of OR", NR"R", NC(O)R"
where each R" is independently H, alkyl, or substituted alkyl.

26. The method of claim 21, corresponding to Formula (XII):
wherein, each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)k R", where k is l, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl; or when more than one R, group is present, two R a may optionality form a cycloalkyl or heterocycloalkyl;
R3 and R4 are independently H, halogen, CN, NO2, alkyl, substituted alkyl, N(R')2, C(O)k R', -C(O)N(R')2, -OR', and -S(O)k R', where k is 1, 2, or 3, where each R' is independently H, alkyl, or substituted alkyl;
X is C, N, or S, with the proviso that when X is O or S, R4 cannot be H, halogen, CN, NO2, alkyl, substituted alkyl, N(R')2, C(O)R', -C(O)N(R')2, -OR', and -S(O)k R'; where k is 1, 2, or 3, and n is 0, 1 or 2.

27. The method of claim 21, corresponding to Formula (XIII):
wherein, each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl;
Y and Z are independently selected from the group consisting of -OH, alkyl substituted oxygen, -SH or alkyl substituted sulfur and when Y and Z taken together can form a cycloalkyl ring.

28. The method of claim 21, corresponding to Formula (XIV):
wherein, each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)k R", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl;
wherein B further comprises -CH=N-O-(alkylene or substituted alkylene)-;
n is 1, 2, or 3; and Y is independently selected from the group consisting of OR", NR"R", NC(O)R"
where each R" is independently H, alkyl, substituted alkyl.

29. The method of claim 15, wherein the compound is reactive with a carbonyl containing agent in aqueous solution under mild conditions.

30. The method of claim 21, the reaction is in an aqueous solution under mild conditions.

31. A method for treating a disorder, condition or disease, the method comprising administering a therapeutically effective amount of an agent having the structure Formula 1, 2, 3, or 4, wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker, linked at one end to an indole-containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(0)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNO2)N(R')-, -N(R')C(NCOOR')N(R')-, -N(R')S(O)k N(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-and each R' is independently II, alkyl, or substituted alkyl;
R1 is H, an amino protecting group, or at least one amino acid; and R2 is OH, an ester protecting group, or at least one amino acid;
n is 0, 1, 2, or 3, and m is 0, 1, 2, or 3, provided that at least one of n or m is not 0;

wherein, each ring in structures 1, 2, 3, and 4 that has an associated R.
group can contain 0, 1, or 2 R, groups and each R, is independently selected from the group consisting. of H, halogen, alkyl, substituted alkyl, -N(R")Z, -C(O)R", -C(O)N(R")2, -OR", and -S(O)k R", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl; or when more than one R a group is present, two R, may optionally form an aryl, cycloalkyl or heterocycloalkyl;.
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
each R5 is independently H, halogen,. alky!, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, OH, NH2, CN, NO2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R", -C(O)2R", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
or R5 is L-X, where, X is a selected from the group consisting of: a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar,group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label;
and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-,-CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, N(R')C(O)O-, -(alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)-S(O)k-(alkylene or substituted alkylene)-S-, -(alkylene or substituted alkylene)-S-S-, -S(O)k N(R')-,-N(R')C(O)N(R')-,-N(R')C(S)N(R')-,-N(R')S(O)k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
when more than one R5 group is present, two ortho R5 groups can optionally form a heterocycloalkyl or an aromatic heterocycloalkyl;
or the -B-indole containing moiety together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable prodrug or solvate thereof.

32. The method of claim 31, further comprising administering a pharmaceutically acceptable carrier.

33. The method of claim 31, wherein X is a polyalkylene oxide.

34. The method of claim 31, wherein X is at least one amino acid.

35. The method of claim 34, wherein the at least one amino acid of X includes a non-natural amino acid.

36. The method of claim 31, wherein X is a detectable label.

37. A method for treating a disorder, condition or disease comprising administering a therapeutically effective amount of an agent having the structure Formula 5, 6, 7, or 8:

wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker, linked at one end to an indole-containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNO2)N(R')-, -N(R')C(NCOOR')N(R')-, -N(R')S(O)k N(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')a-N=N-, and -C(R')2-N(R')-N(R')-and each R' is independently H, alkyl, or substituted alkyl;
R1 is H, an amino protecting group, or at least one amino acid; and R2 is OH, an ester protecting group, or at least one amino acid;
n is 0, 1, 2, or 3, and m is 0, 1, 2,or 3,provided that at least one of n or m is not 0;
wherein, each ring in structures 1, 2, 3, and 4 that has an associated R a group can contain.0, 1, or 2 R a groups and each R a is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR'", and -S(O)k R", where k is 1, 2, or 3, where each R" is independently H alkyl, or substituted alkyl; or when more than one R a group is present, two R a may optionally form an aryl, cycloalkyl or heterocycloalkyl;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
each R5 is independently H, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, OH, NH2, CN, NO2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R", -C(O)2R", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
or R5 is L-X, where, X is a selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene,glycol; a photocrosslinker;at least one amino acid; at least one:sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label;
and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0-(alkylene or substituted alkylene)-, -S-,-S-(alkylene or substituted allylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-,-CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -(alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)-S(O)k-( alkylene or substituted alkylene)-S-, -(alkylene.or substituted alkylene)-S-S-, -S(O)k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
when more than one R5 group is present, two ortho R5 groups can optionally form a heterocycloalkyl or an aromatic heterocycloalkyl;
or the -B-indole containing moiety together form a bicyclic or tricyclic.cycloalkyl or heterocycloalkyl comprising at least one, indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable prodrug or solvate thereof.

38. The method of claim 37, further comprising administering a pharmaceutically acceptable carrier.

39. The method of claim 37, wherein R, and R2 are both at least one amino acid.

40. The method of claim 37, wherein X is a polyalkylene oxide.

41. The method of claim 37, wherein X is at least one amino acid.

42. A method for detecting the presence of a polypeptide in a patient, the method comprising administering an effective amount of a homologous non-natural amino acid polypeptide comprising at least one non-natural amino acid having the structures of compounds 1-4:

wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, substituted alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when,present is a linker, linked at one end to an indole-containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-where k is 1,2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NN02)N(R')-, -N(R')C(NCOOR')N(R')-, -N(R')S(O)k N(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-and each R' is independently H, alkyl, or substituted alkyl;
R1 is H, an amino protecting group, or at least one amino acid; and R2 is OH, an ester protecting group, or at least one amino acid;
n is 0, 1, 2, or 3, and m is 0, 1, 2, or 3, provided that at least one of n or m is not 0;

wherein, each ring in structures 1,2,3, and 4 that has an associated R a,group can contain 0, 1, or 2 R a groups and each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)k R", where k is 1, 2, or 3, where each R" is independently H, alkyl, or substituted alkyl; or when more than one R a group is present, two R a may optionally form an aryl, cycloalkyl or heterocycloalkyl;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
each R5 is independently H, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, OH, NH2, CN, NO2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R", -C(O)2R", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
or R5 is L-X, where, X is a selected from the group consisting of: a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label;
and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-,-CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -(alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(alkylene or substituted alkylene)-,-(alkylene or substituted alkylene)-S(O)k-(alkylene or substituted alkylene)-S-, -(alkylene or substituted alkylene)-S-S-, -S(O)k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;
when more than one R5 group is present, two ortho R5 groups can optionally form a heterocycloalkyl or an aromatic heterocycloalkyl;
or the -B-indole containing moiety together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable prodrug or solvate thereof.

43. The method of claim 42, wherein the polypeptide is a protein homologous to a therapeutic protein selected from the group consisting of: alpha-1 antitrypsin, angiostatin, antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C
chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF
receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF),. tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.